Cytokine antagonist molecules

ABSTRACT

This invention relates to a novel protein (INSP052), herein identified as an immunoglobulin domain-containing cell surface recognition molecule and to the use of this proteins and nucleic acid sequences from the encoding gene in the diagnosis, prevention and treatment of disease. The invention also relates to the identification of the extracellular domain of INSP052.

REFERNCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of InternationalApplication PCT/GB03/01851 filed on Apr. 30, 2003 designating the U.S.,which claims priority from Great Britain Application GB 0209884.6 filedApr. 30, 2002.

[0002] Each of the foregoing applications, and each document cited orreferenced in each of the foregoing applications, including during theprosecution of each of the foregoing applications and (“applicationcited documents”), and any manufacturer 's instructions or cataloguesfor any products cited or mentioned in each of the foregoingapplications and articles and in any of the application cited documents,are hereby incorporated herein by reference. Furthermore, all documentscited in this text, and all documents cited or referenced in documentscited in this text, and any manufacturer's instructions or cataloguesfor any products cited or mentioned in this text or in any documenthereby incorporated into this text, are hereby incorporated herein byreference. Documents incorporated by reference into this text or anyteachings therein may be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

[0003] It is noted that in this disclosure and particularly in theclaims, terms such as “comprises”, “comprised”, “comprising” and thelike can have the meaning attributed to it in U.S. Patent law; e.g.,they can mean “includes”, “included”, “including”, and the like; andthat terms such as “consisting essentially of” and “consists essentiallyof” have the meaning ascribed to them in U.S. Patent law, e.g., theyallow for elements not explicitly recited, but exclude elements that arefound in the prior art or that affect a basic or novel characteristic ofthe invention.

[0004] All publications, patents and patent applications cited hereinare incorporated in full by reference.

SUMMARY OF THE INVENTION

[0005] This invention relates to novel proteins (termed INSP052 andINSP055), herein identified as immunoglobulin domain-containing cellsurface recognition molecules and to the use of these proteins andnucleic acid sequences from the encoding genes in the diagnosis,prevention and treatment of disease, for instance in the diagnosis,prevention and treatment of inflammatory diseases, auto-immune diseases,liver disease or liver failure.

BACKGROUND

[0006] The process of drug discovery is presently undergoing afundamental revolution as the era of functional genomics comes of age.The term “functional genomics” applies to an approach utilisingbioinformatics tools to ascribe function to protein sequences ofinterest. Such tools are becoming increasingly necessary as the speed ofgeneration of sequence data is rapidly outpacing the ability of researchlaboratories to assign functions to these protein sequences.

[0007] As bioinformatics tools increase in potency and in accuracy,these tools are rapidly replacing the conventional techniques ofbiochemical characterisation. Indeed, the advanced bioinformatics toolsused in identifying the present invention are now capable of outputtingresults in which a high degree of confidence can be placed.

[0008] Various institutions and commercial organisations are examiningsequence data as they become available and significant discoveries arebeing made on an on-going basis. However, there remains a continuingneed to identify and characterise further genes and the polypeptidesthat they encode, as targets for research and for drug discovery.

[0009] Recently, a remarkable tool for the evaluation of sequences ofunknown function has been developed by the Applicant for the presentinvention. This tool is a database system, termed the Biopendium searchdatabase, that is the subject of WO01/69507. This database systemconsists of an integrated data resource created using proprietarytechnology and containing information generated from an all-by-allcomparison of all available protein or nucleic acid sequences.

[0010] The aim behind the integration of these sequence data fromseparate data resources is to combine as much data as possible, relatingboth to the sequences themselves and to information relevant to eachsequence, into one integrated resource. All the available data relatingto each sequence, including data on the three-dimensional structure ofthe encoded protein, if this is available, are integrated together tomake best use of the information that is known about each sequence andthus to allow the most educated predictions to be made from comparisonsof these sequences. The annotation that is generated in the database andwhich accompanies each sequence entry imparts a biologically relevantcontext to the sequence information.

[0011] This data resource has made possible the accurate prediction ofprotein function from sequence alone. Using conventional technology,this is only possible for proteins that exhibit a high degree ofsequence identity (above about 20%-30% identity) to other proteins inthe same functional family. Accurate predictions are not possible forproteins that exhibit a very low degree of sequence homology to otherrelated proteins of known function.

[0012] Signal Peptide-Containing Proteins

[0013] The ability of cells to make and secrete extracellular proteinsis central to many biological processes. Enzymes, growth factors,extracellular matrix proteins and signaling molecules are all secretedby cells. This is through fusion of a secretory vesicle with the plasmamembrane. In most cases, but not all, proteins are directed to theendoplasmic reticulum and into secretory vesicles by a signal peptide.Signal peptides are cis-acting sequences that affect the transport ofpolypeptide chains from the cytoplasm to a membrane bound compartmentsuch as a secretory vesicle. Polypeptides that are targeted to thesecretory vesicles are either secreted into the extracellular matrix orare retained in the plasma membrane. The polypeptides that are retainedin the plasma membrane will have one or more transmembrane domains.Examples of signal peptide containing proteins that play a central rolein the functioning of a cell are cytokines, hormones, extracellularmatrix proteins, adhesion molecules, receptors, proteases, and growthand differentiation factors.

[0014] Immunoglobulin Domain-Containing Cell Surface RecognitionMolecules

[0015] Immunoglobulin domain-containing cell surface recognitionmolecules have been shown to play a role in diverse physiologicalfunctions, many of which can play a role in disease processes.Alteration of their activity is a means to alter the disease phenotypeand as such identification of novel immunoglobulin domain-containingcell surface recognition molecules is highly relevant as they may play arole in many diseases, particularly inflammatory disease, oncology, andcardiovascular disease. Immunoglobulin domain-containing cell surfacerecognition molecules are involved in a range of biological processes,including: embryogenesis (Martin-Bermudo, M. D. et al, Development. 2000127(12):2607-15; Chen, L. M., et al., J. Neurosci. 2000 20(10):3776-84;Zweegman, S., et al, Exp Hematol. 2000 28(4):401-10; Darribere, T., etal., Biol Cell. 2000 92(1):5-25), maintenance of tissue integrity(Eckes, B., et al., J Cell Sci. 2000 113(Pt 13):2455-2462; Buckwalter,J. A., et al., Instr Course Lect. 2000 49:481-9; Frenette, P. S., etal., J Exp Med. 2000 191(8):1413-22; Delmas, V., et al, Dev Biol. 1999216(2):491-506; Humphries, M. J., et al., Trends Pharmacol Sci. 200021(1):29-32; Miosge, N., et al, Lab Invest. 1999 79(12):1591-9; NagaokaT, et al. Am J Pathol 2000 July 157:1 237-47; Nwariaku F E, et al. JTrauma 1995 39(2): 285-8; Zhu X, et al. Zhonghua Zheng Xing Shao ShangWai Ke Za Zhi 1999 15(1): 53-5), leukocyte extravasation/inflammation(Lim, L. H., et al. Am J Respir Cell Mol. Biol. 2000 22(6):693-701;Johnston, B., et al., Microcirculation. 2000 7(2):109-18; Mertens, A.V., et al., Clin Exp Allergy. 1993 23(10):868-73; Chcialowski, A., etal., Pol Merkuriusz Lek. 2000 7(43):13-7; Rojas, A. I., et al, Crit RevOral Biol Med. 1999 10(3):337-58; Marinova-Mutafchieva, L., et al.,Arthritis Rheum. 2000 43(3):638-44; Vijayan, K. V., et al, J ClinInvest. 2000 105(6):793-802; Currie, A. J., et al,. J. Immunol. 2000164(7):3878-86; Rowin, M. E., et al., Inflammation. 2000 24(2):157-73;Johnston, B., et al., J. Immunol. 2000 164(6):3337-44; Gerst, J. L., etal., J Neurosci Res. 2000 59(5):680-4; Kagawa, T. F., et al., Proc NatlAcad Sci USA. 2000 97(5):2235-40; Hillan, K. J., et al., Liver. 199919(6):509-18; Panes, J., 1999 22(10):514-24; Arao, T., et al., J ClinEndocrinol Metab. 2000 85(1):382-9; Souza, H. S., et al., Gut. 199945(6):856-63; Grunstein, M. M., et al., Am J Physiol Lung Cell MolPhysiol. 2000 278(6):L1 154-63; Mertens, A. V., et al., Clin ExpAllergy. 1993 23(10):868-73; Berends, C., et al., Clin Exp Allergy. 199323(11):926-33; Femvik, E., et al., Inflammation. 2000 24(1):73-87;Bocchino, V., et al., J Allergy Clin Immunol. 2000 105(1 Pt 1):65-70;Jones S C, et al, Gut 1995 36(5):724-30; Liu C M, et al, Ann AllergyAsthma Immunol 1998 81(2):176-80; McMurray R W Semin Arthritis Rheum1996 25(4):215-33; Takahashi H, et al Eur J Immunol 1992 22(11):2879-85; Carlos T, et al J Heart Lung Transplant 1992 11(6): 1103-8;Fabrega E, et al, Transplantation 2000 69(4): 569-73; Zohrens G, et al,Hepatology 1993 18(4): 798-802; Montefort S, et al. Am J Respir CritCare Med 1994 149(5): 1149-52), oncogenesis (Orr, F. W., et al., Cancer.2000 88(S12):2912-2918; Zeller, W., et al., J Hematother Stem Cell Res.1999 8(5):539-46; Okada, T., et al., Clin Exp Metastasis. 199917(7):623-9; Mateo, V., et al., Nat Med. 1999 5(11):1277-84; Yamaguchi,K., et al., J Exp Clin Cancer Res. 2000 19(1):113-20; Maeshima, Y., etal., J Biol. Chem. 2000 275(28):21340-8; Van Waes, C., et al., Int JOncol. 2000 16(6):1189-95; Damiano, J. S., et al., Leuk Lymphoma. 200038(1-2):71-81; Seflor, R. E., et al., Cancer Metastasis Rev. 199918(3):359-75; Shaw, L. M., J Mammary Gland Biol Neoplasia. 19994(4):367-76; Weyant, M. J., et al., Clin Cancer Res. 2000 6(3):949-56),angiogenesis (Koch A E, et al Nature 1995 376 (6540): 517-9; Wagener C &Ergun S. Exp Cell Res 2000 261(1): 19-24; Ergun S, et al. Mol Cell 20005(2): 311-20), bone resorption (Hartman G D, & Duggan M E. Expert OpinInvestig Drugs 2000 9(6): 1281-91; Tanaka Y, et al. J Bone Miner Res1995 10(10): 1462-9; Lark M W, et al. J Pharmacol Exp Ther 1999 291(2):612-7; Raynal C, et al. Endocrinology 1996 137(6):2347-54; Ilvesaro J M,et al. Exp Cell Res 1998 242(1): 75-83), neurological dysfunction(Ossege L M, et al. Int Immunopharmacol 2001 1:1085-100; Bitsch A, etal, Stroke 1998 29:2129-35; Iadecola C & Alexander M. Curr Opin Neurol2001 14:89-94; Becker K, et al Stroke 2001 32(1): 206-11; Relton J K, etal Stroke 2001 32(1): 199-205; Hamada Y, et al J Neurochem 199666:1525-31), thrombogenesis (Wang, Y. G., et al., J Physiol (Lond). 2000526(Pt 1):57-68; Matsuno, H., et al., Nippon Yakurigaku Zasshi. 2000115(3):143-50; Eliceiri, B. P., et al., Cancer J Sci Am. 2000 6(Suppl3):S245-9; von Beckerath, N., et al., Blood. 2000 95(11):3297-301;Topol, E. J., et al., Am Heart J. 2000 139(6):927-33; Kroll, H., et al.,Thromb Haemost. 2000 83(3):392-6), and invasion/adherence of bacterialpathogens to the host cell (Dersch P, et al. EMBO J 1999 18(5):1199-1213).

[0016] The detailed characterisation of the structure and function ofseveral immunoglobulin-domain containing cell surface recognitionmolecule families has led to active programs by a number ofpharmaceutical companies to develop modulators for use in the treatmentof diseases involving inflammation, oncology, neurology, immunology andcardiovascular function. Immunoglobulin domain containing cell surfacerecognition molecules are involved in virtually every aspect of biologyfrom embryogenesis to apoptosis. They are essential to the structuralintegrity and homeostatic functioning of most tissues. It is thereforenot surprising that defects in immunoglobulin domain containing cellsurface recognition molecules cause disease and that many diseasesinvolve modulation of immunoglobulin domain containing cell surfacerecognition molecule function. The members of this family are describedbelow in Table 1.

[0017] The Immunoglobulin domain containing cell surface recognitionmolecule family in fact contains several distinct families. Of thesefamilies, some are of particular pharmaceutical interest due to smallmolecule tractibility. They include:

[0018] 1. The immunoglobulin adhesion molecules represent the counterreceptors for the integrins and includes the intracellular adhesionmolecules (ICAMs) and vascular cell adhesion molecules (VCAMs). Membersare composed of variable numbers of globular, immunoglobulin-like,extracellular domains. Some members of the family, for example, PECAM-1(CD31) and NCAM, mediate homotypic adhesion. Some members of the family,for example ICAM-1 and VCAM-1, mediate adhesion via interactions withintegrins.

[0019] 2. Cell surface growth factor receptors. Growth factors areextracellular and in order to exert a biological effect they interactwith specific, high affinity receptors located on the plasma membranesof target cells. The molecular characterisation of a variety ofdifferent growth factor receptors revealed that they fall into definedfamilies; the tyrosine kinase receptors, G-protein associated seventransmembrane receptors, and the serine/threonine kinase receptors. Thetyrosine kinase receptors are characterised by an extracellular domain,a transmembrane domain, and an intracellular domain which possesstyrosine kinase activity. VEGFR, PDGFR, FGFR, CSF-1R and c-KIT areexamples of tyrosine kinase growth factor receptors which also containimmunoglobulin domains in the extracellular portion. Dys-regulation ofgrowth factor function results in many different disease phenotypes,including, but not exclusive to oncology (Bartucci M et al, (2001)Cancer Res. September 15;61(18):6747-54, Dias S et al., (2001) Proc NatlAcad Sci USA. September 11;98(19):10857-62, Djavan B et al., (2001)World J Urol. 19(4):225-33), inflammation (Fiocchi C. (2001) J ClinInvest. August;108(4):523-6, Hodge S et al., (2001) Respirology.September;6(3):205-211, Fenwick S A et al., (2001) J Anat.September;199(Pt 3):231-40), neurological (Cooper J D et al., (2001)Proc Natl Acad Sci USA 98(18):10439-44, Fahnestock M et al, (2001) MolCell Neurosci 18(2):210-20), and metabolism (Vickers M H et al., (2001)Endocrinology. 142(9):3964-73). TABLE 1 Immunoglubulin domain-containingcell surface recognition molecules Receptor Ligand Distribution ICAM-1LFA-1 (CD11a/CD18) Widespread, endothelial cells, fibroblasts, 5 Igdomains Mac-1 (CD11b/CD18), epithelium, monocytes, lymphocytes,dendritic CD43 cells, chondrocytes. ICAM-2 LFA-1 (CD11b) endothelialcells (high): lymphocytes, monocytes, 2 Ig domains basophils, platelets(low). ICAM-3 LFA-1 (αd/CD 18) Lymphocytes, monocytes, neutrophils,eosinophils, 5 Ig domains basophils. VCAM-1 α4β1, α4β7 Endothelialcells, monocytes, fibroblasts, dendritic 6 or 7 Ig cells, bone marrowstromal cells, myoblasts. domains LFA-3 CD2 Endothelial cells,leukocytes, epithelial cells 6 Ig domains PECAM-1 CD31, heparinEndothelial cells (at EC-EC junctions), T cell (CD31) subsets,platelets, neutrophils, eosinophils, monocytes, smooth muscle cells,bone marrow stem cells. NCAM NCAM, heparin SO₄ Neural cells, muscleMAdCAM-1 α4β7, L-selectin Peyer's patch, mesenteric lymph nodes, mucosal4 Ig domains endothelial cells, spleen. CD2 CD58, CD59, CD48 Tlymphocytes VEGFR VEGF Widespread, retina, umbilical vein, adrenal, NT2neuronal precursor cells FGFR FGF Widespread, brain, colon, ovary KITStem Cell Factor, MGF Widespread, foetus, melanocytes, gall bladder,cerebellum, gastric epithelium (low) PDGFR PDGF Widespread, breast,placenta, fibroblast, lung, ovary, skin, heart CSF-1R CSF Widespread,placenta, liver, multiple sclerosis lesions, spleen, lung, breast.

[0020] Immunoglobulin domain-containing cell surface recognitionmolecules have thus been shown to play a role in diverse physiologicalfunctions, many of which can play a role in disease processes.Alteration of their activity is a means to alter the disease phenotypeand as such identification of novel Immunoglobulin domain-containingcell surface recognition molecules is highly relevant as they may play arole in many diseases, particularly immunology, inflammatory disease,oncology, cardiovascular disease, central nervous system disorders andinfection.

THE INVENTION

[0021] The invention is based on the discovery that the INSP052 andINSP055 proteins function as immunoglobulin domain-containing cellsurface recognition molecules. Examples of immunoglobulindomain-containing cell surface recognition molecules are listed in Table1.

[0022] In one embodiment of the first aspect of the invention, there isprovided a polypeptide which:

[0023] (i) comprises or consists of the amino acid sequence as recitedin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, or SEQ ID NO:26;

[0024] (ii) is a fragment thereof having the activity of a polypeptideaccording to (i), or having an antigenic determinant in common with apolypeptide according to (i); or

[0025] (iii) is a functional equivalent of (i) or (ii).

[0026] By “the activity of a polypeptide according to (i)”, we refer toimmunoglobulin domain-containing cell surface recognition moleculeactivity. By immunoglobulin domain-containing cell surface recognitionmolecule activity we refer to polypeptides that comprise amino acidsequence or structural features that can be identified as conservedfeatures within the immunoglobulin domain-containing cell surfacerecognition molecule family. Included within this definition is activityas a cytokine antagonist, particularly as an antagonist of cytokineexpression and/or secretion, particularly with respect to TNF-alpha,IL-4 and/or IL-2.

[0027] Evidence is presented in the Examples section below that theextracellular domain of INSP052 (also referred to herein as INSP052EC)downregulates TNF-alpha, IL-4 and IL-2 secretion in vitro in aConcanavalin A (ConA) stimulated human peripheral blood mononuclearcells (hPBMC) assay. In addition, delivery of INSP052EC cDNA in an invivo model of fulminant hepatitis was found to decrease TNF-alpha andm-IL-6 levels in serum and had a significant effect on the reduction oftransaminases measured in serum. This effect was confirmed bysubcutaneous INSP052EC protein injections.

[0028] The decrease in aspartate aminotransferase (ASAT) and alanineaminotransferase (ALAT) levels noted might be due to both decreasedTNF-alpha and IL-4 levels. TNF-alpha and IL-4 are important cytokinesinvolved in liver damage induced after ConA injection. In this mousemodel of liver hepatitis, TNF-alpha is mainly produced by hepaticmacrophages, the so-called Kupfer cells, whereas IL-4 is produced byliver (natural killer T) NKT cells. Anti TNF-alpha antibodies have beenshown to confer protection against disease (Seino et al. 2001, Annals ofsurgery 234, 681) and inhibition of IL-4 production by NKT cells wasshown to be hepato-protective in T-cell mediated hepatitis in mouse(Ajuebor et al. 2003 J. Immunology 170, 5252-9). Accordingly, it isconsidered that INSP052, INSP052EC (SEQ ID NO.20 and SEQ ID NO.22) andrelated functionally equivalent proteins will be useful in treatingauto-immune, viral or acute liver diseases as well as alcoholic liverfailures. They are likely also to be effective in treating otherinflammatory diseases.

[0029] The polypeptide having the sequence recited in SEQ ID NO:2 isreferred to hereafter as “the INSP052 exon 1 polypeptide”. Thepolypeptide having the sequence recited in SEQ ID NO:4 is referred tohereafter as “the INSP052 exon 2 polypeptide”. The polypeptide havingthe sequence recited in SEQ ID NO:6 is referred to hereafter as “theINSP052 exon 3 polypeptide”. The polypeptide having the sequence recitedin SEQ ID NO:8 is referred to hereafter as “the INSP052 exon 4polypeptide”. The polypeptide having the sequence recited in SEQ IDNO:10 is referred to hereafter as “the INSP052 exon 5 polypeptide”. Thepolypeptide having the sequence recited in SEQ ID NO:12 is referred tohereafter as “the INSP052 exon 6 polypeptide”. The polypeptide havingthe sequence recited in SEQ ID NO:14 is referred to hereafter as “theINSP052 exon 7 polypeptide”. Combining SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14 producesthe sequence recited in SEQ ID NO:16. The polypeptide having thesequence recited in SEQ ID NO:16 is referred to hereafter as the INSP052polypeptide. The polypeptide having the sequence recited in SEQ ID NO:20is the extracellular domain of INSP052. The polypeptide having thesequence recited in SEQ ID NO:22 is referred to hereafter as theextracellular domain of the mature INSP052 polypeptide. The polypeptidehaving the sequence recited in SEQ ID NO:24 is referred to hereafter asthe mature INSP052 exon 2 polypeptide. The polypeptide having thesequence recited in SEQ ID NO:26 is referred to hereafter as the matureINSP052 polypeptide.

[0030] The term “INSP052 exon polypeptides” as used herein includespolypeptides comprising or consisting of the polypeptide sequences setforth herein, including the INSP052 exon 1 polypeptide, the INSP052 exon2 polypeptide, the INSP052 exon 3 polypeptide, the INSP052 exon 4polypeptide, the INSP052 exon 5 polypeptide, the INSP052 exon 6polypeptide, the INSP052 exon 7 polypeptide, the INSP052 polypeptide,the extracellular domain of INSP052, the extracellular domain of matureINSP052, the INSP052 mature exon 2 polypeptide, and the mature INSP052polypeptide.

[0031] In one embodiment, the polypeptide according to this embodimentconsists of the amino acid sequence recited in SEQ ID NO:16 or is afragment of or functional equivalent thereof. In another embodiment, thepolypeptide consists of the amino acid sequence recited in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, orSEQ ID NO:14, or a variant thereof.

[0032] In a further embodiment of the first aspect of the inventionthere is provided a polypeptide which:

[0033] i) comprises or consists of the amino acid sequence as recited inSEQ ID NO:20 or SEQ ID NO:22;

[0034] ii) is a fragment thereof having the activity of a polypeptideaccording to (i), or having an antigenic determinant in common with apolypeptide according to (i); or

[0035] iii) is a functional equivalent of (i) or (ii).

[0036] The amino acid sequence recited in SEQ ID NO:20 represents theextracellular domain of INSP052 and corresponds to amino acids 1-240 ofthe full length protein (see the Examples section). SEQ ID NO:22represents the extracellular domain of mature INSP052. See also FIG. 7for the extracellular domain of INSP052.

[0037] It is considered highly likely that the extracellular domain willfold correctly and show biological activity if additional residues Cterminal and/or N terminal of these boundaries in the polypeptidesequence are included in the polypeptide fragment. For example, anadditional 5, 10, 20, 30, 40, 50 or even 100 amino acid residues fromthe INSP052 polypeptide sequence, or from a homologous sequence, may beincluded at either or both the C terminal and/or N terminal of theboundaries of the receptor binding domain, without prejudicing theability of the polypeptide fragment to fold correctly and exhibitbiological activity. Extensions as large as 100 or 200 residues may benecessary due to the presence of large loops between secondarystructural elements.

[0038] For truncated variants of the INSP052 extracellular domain, oneor a few amino acid residues (for example, 2, 3, 4, 5, 10, 15, 20, 25,30 or more) may be deleted at either or both the C terminus or the Nterminus of the domain without prejudicing biological activity.

[0039] As discussed below, the polypeptides of the invention may beprovided in the form of a fusion protein or as “free-standing” protein.Accordingly, one embodiment of the invention provides a polypeptidewhich consists of the extracellular domain of INSP052. Anotherembodiment of the invention provides a polypeptide which consists ofINSP052 (the full length protein or the extracellular domain thereof,including the mature versions thereof) fused with at least one otherpolypeptide to form a fusion protein.

[0040] In a second embodiment of the first aspect of the invention,there is provided a polypeptide which:

[0041] (i) comprises or consists of the amino acid sequence as recitedin SEQ ID NO: 18,

[0042] (ii) is a fragment thereof having the activity of a polypeptideof (i), or having an antigenic determinant in common with a polypeptideof (i); or

[0043] (iii) is a functional equivalent of (i) or (ii).

[0044] By “the activity of a polypeptide according to (i)”, we refer toimmunoglobulin domain-containing cell surface recognition moleculeactivity.

[0045] Preferably, the polypeptide according to this embodiment consistsof the amino acid sequence recited in SEQ ID NO:18 or is a fragment ofor functional equivalent thereof.

[0046] The polypeptide having the sequence recited in SEQ ID NO:18 isreferred to hereafter as “the INSP055 polypeptide”.

[0047] In a second aspect, the invention provides a purified nucleicacid molecule which encodes a polypeptide of the first aspect of theinvention.

[0048] Preferably, the purified nucleic acid molecule comprises orconsists of the nucleic acid sequence as recited in SEQ ID NO:1(encoding the INSP052 exon 1 polypeptide), SEQ ID NO:3 (encoding theINSP052 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP052 exon 3polypeptide), SEQ ID NO:7 (encoding the INSP052 exon 4 polypeptide), SEQID NO:9 (encoding the INSP052 exon 5 polypeptide), SEQ ID NO:11(encoding the INSP052 exon 6 polypeptide), SEQ ID NO:13 (encoding theINSP052 exon 7 polypeptide), SEQ ID NO:15 (encoding the INSP052polypeptide), SEQ ID NO:17 (encoding the INSP055 polypeptide), SEQ IDNO:20 (encoding the extracellular domain of the INSP052 polypeptide),SEQ ID NO:22 (encoding the extracellular domain of the INSP052 maturepolypeptide), SEQ ID NO:24 (encoding the mature INSP052 exon 2polypeptide), SEQ ID NO:26 (encoding the mature INSP052 polypeptide) oris a redundant equivalent or fragment of any one of these sequences.

[0049] Combining the sequences recited in SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13produces the sequence recited in SEQ ID NO:15.

[0050] Combining the sequences recited in SEQ ID NO:23, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13 produces thesequence recited in SEQ ID NO:25.

[0051] In one embodiment of the second aspect of the invention there isprovided a nucleic acid molecule which encodes a polypeptide whichcomprises or consists of the extracellular domain of INSP052 (SEQ IDNO:20). Preferably, the nucleic acid molecule comprises or consists ofthe nucleic acid sequence set forth in SEQ ID NO:19. This is also setout in FIG. 7, although these sequences include histidine residues addedto the C terminal.

[0052] In one embodiment of the second aspect of the invention there isprovided a nucleic acid molecule which encodes a polypeptide whichcomprises or consists of the extracellular domain of mature INSP052 (SEQID NO:22). Preferably, the nucleic acid molecule comprises or consistsof the nucleic acid sequence set forth in SEQ ID NO:21. This is also setout in FIG. 7, although these sequences include histidine residues addedto the C terminal.

[0053] In a third aspect, the invention provides a purified nucleic acidmolecule which hybridizes under high stringency conditions with anucleic acid molecule of the second aspect of the invention.

[0054] In a fourth aspect, the invention provides a vector, such as anexpression vector, that contains a nucleic acid molecule of the secondor third aspect of the invention.

[0055] In a fifth aspect, the invention provides a host cell transformedwith a vector of the fourth aspect of the invention.

[0056] In a sixth aspect, the invention provides a ligand which bindsspecifically to, and which preferably inhibits the activity of apolypeptide of the first aspect of the invention.

[0057] By “the activity of a polypeptide of the invention” and similarexpressions, we refer to activity characteristic of immunoglobulindomain-containing cell surface recognition molecules. In particular,included within this definition is activity as a cytokine antagonist,particularly as an antagonist of cytokine expression and/or secretion,particularly with respect to TNF-alpha, IL-4 and IL-2.

[0058] In a seventh aspect, the invention provides a compound that iseffective to alter the expression of a natural gene which encodes apolypeptide of the first aspect of the invention or to regulate theactivity of a polypeptide of the first aspect of the invention.

[0059] A compound of the seventh aspect of the invention may eitherincrease (agonise) or decrease (antagonise) the level of expression ofthe gene or the activity of the polypeptide.

[0060] Importantly, the identification of the function of the INSP052and INSP055 polypeptides allows for the design of screening methodscapable of identifying compounds that are effective in the treatmentand/or diagnosis of disease. Ligands and compounds according to thesixth and seventh aspects of the invention may be identified using suchmethods. These methods are included as aspects of the present invention.

[0061] Evidence is presented in the Examples section below that theextracellular domain of INSP052 may be used to prevent or treatinflammatory diseases, auto-immune diseases, liver disease or liverfailure. Accordingly, the provision of a compound according to theseventh aspect of the invention which mimics extracellular domain ofINSP052 conformationally, or is an agonist of the extracellular domainof INSP052 is particularly preferred since such a compound may findutility in the prevention or treatment of an inflammatory disease, anauto-immune disease, liver disease or liver failure as described above.

[0062] In an eighth aspect, the invention provides a polypeptide of thefirst aspect of the invention, or a nucleic acid molecule of the secondor third aspect of the invention, or a vector of the fourth aspect ofthe invention, or a host cell of the fifth aspect, or a ligand of thesixth aspect of the invention, or a compound of the seventh aspect ofthe invention, for use in therapy or diagnosis, preferably in relationto inflammatory diseases, auto-immune diseases, liver disease (includingviral or acute liver disease) and liver failure (including alcoholicliver failure).

[0063] The moieties of the first, second, third, fourth, fifth and sixthaspects of the invention may also be used in the manufacture of amedicament for the prevention or treatment of diseases including, butnot limited to, cell proliferative disorders, autoimmune/inflammatorydisorders, cardiovascular disorders, neurological and psychiatricdisorders, developmental disorders, genetic disorders, metabolicdisorders, infections and other pathological conditions.

[0064] These diseases preferably include neoplasm, cancer, brain tumour,glioma, bone tumor, lung tumor, breast tumour, prostate tumour, colontumour, hemangioma, myeloproliferative disorder, leukemia, hematologicaldisease, neutropenia, thrombocytopenia, angiogenesis disorders,dermatological disease, ageing, wounds, burns, fibrosis, cardiovasculardisease, restensosis, heart disease, peripheral vascular disease,coronary artery disease, oedema, thromboembolism, dysmenorrhea,endometriosis, pre-eclampsia, lung disease, COPD, asthma bone disease,renal disease, glomerulonephritis, liver disease, Crohn's disease,gastritis, ulcerative colitis, ulcer, immune disorder, autoimmunedisease, arthritis, rheumatoid arthritis, psoriasis, epidermolysisbullosa, systemic lupus erythematosus, ankylosing spondylitis, Lymedisease, multiple sclerosis, neurodegeneration, stroke, brain/spinalcord injury, Alzheimer's disease, Parkinson's disease, motor neuronedisease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection,fungal infection, ocular disorder, macular degeneration, glaucoma,diabetic retinopathy, ocular hypertension and other conditions in whichimmunoglobulin domain containing cell surface recognition molecules areimplicated.

[0065] It is particularly preferred that the moieties of the first,second, third, fourth, fifth and sixth aspects of the invention are usedin the manufacture of a medicament for the treatment of inflammatorydiseases, auto-immune diseases, liver disease (including viral or acuteliver disease) and liver failure (including alcoholic liver failure).

[0066] In a ninth aspect, the invention provides a method of diagnosinga disease in a patient, comprising assessing the level of expression ofa natural gene encoding a polypeptide of the first aspect of theinvention or the activity of a polypeptide of the first aspect of theinvention in tissue from said patient and comparing said level ofexpression or activity to a control level, wherein a level that isdifferent to said control level is indicative of disease. Such a methodwill preferably be carried out in vitro.

[0067] Similar methods may be used for monitoring the therapeutictreatment of disease in a patient, wherein altering the level ofexpression or activity of a polypeptide or nucleic acid molecule overthe period of time towards a control level is indicative of regressionof disease.

[0068] A preferred method for detecting polypeptides of the first aspectof the invention comprises the steps of: (a) contacting a ligand, suchas an antibody, of the sixth aspect of the invention with a biologicalsample under conditions suitable for the formation of aligand-polypeptide complex; and (b) detecting said complex.

[0069] A number of different methods according to the ninth aspect ofthe invention exist, as the skilled reader will be aware, such asmethods of nucleic acid hybridization with short probes, point mutationanalysis, polymerase chain reaction (PCR) amplification and methodsusing antibodies to detect aberrant protein levels. Similar methods maybe used on a short or long term basis to allow therapeutic treatment ofa disease to be monitored in a patient. The invention also provides kitsthat are useful in these methods for diagnosing disease.

[0070] Preferably, the disease diagnosed by a method of the ninth aspectof the invention is a disease in which immunoglobulin domain-containingcell surface recognition molecules are implicated, as described above.

[0071] A preferred disease diagnosed by a method of the ninth aspect ofthe invention is an inflammatory disease, auto-immune disease, liverdisease (including viral or acute liver disease) or liver failure(including alcoholic liver failure).

[0072] In a tenth aspect, the invention provides for the use of thepolypeptides of the first aspect of the invention as immunoglobulindomain-containing cell surface recognition molecules. The importance ofthe Ig domain in cell surface receptors is described in Lokker N A etal., “Functional importance of platelet-derived growth factor (PDGF)receptor extracellular immunoglobulin-like domains. Identification ofPDGF binding site and neutralizing monoclonal antibodies,” J Biol Chem1997 December. 26;272(52):33037-44.

[0073] The invention also provides for the use of a nucleic acidmolecule according to the second or third aspects of the invention toexpress a protein that possesses immunoglobulin domain-containing cellsurface recognition molecule activity. The invention also provides amethod for effecting immunoglobulin domain-containing cell surfacerecognition molecule activity, said method utilising a polypeptide ofthe first aspect of the invention.

[0074] In an eleventh aspect, the invention provides a pharmaceuticalcomposition comprising a polypeptide of the first aspect of theinvention, or a nucleic acid molecule of the second or third aspect ofthe invention, or a vector of the fourth aspect of the invention, or ahost cell of the fifth aspect of the invention, or a ligand of the sixthaspect of the invention, or a compound of the seventh aspect of theinvention, in conjunction with a pharmaceutically-acceptable carrier.

[0075] In a twelfth aspect, the present invention provides a polypeptideof the first aspect of the invention, or a nucleic acid molecule of thesecond or third aspect of the invention, or a vector of the fourthaspect of the invention, or a host cell of the fifth aspect of theinvention, or a ligand of the sixth aspect of the invention, or acompound of the seventh aspect of the invention, for use in therapy ordiagnosis. These molecules may also be used in the manufacture of amedicament for the treatment of diseases including, but not limited to,cell proliferative disorders, autoimmune/inflammatory disorders,cardiovascular disorders, neurological and psychiatric disorders,developmental disorders, genetic disorders, metabolic disorders,infections and other pathological conditions. These diseases preferablyinclude neoplasm, cancer, brain tumour, glioma, bone tumor, lung tumor,breast tumour, prostate tumour, colon tumour, hemangioma,myeloproliferative disorder, leukemia, hematological disease,neutropenia, thrombocytopenia, angiogenesis disorders, dermatologicaldisease, ageing, wounds, burns, fibrosis, cardiovascular disease,restensosis, heart disease, peripheral vascular disease, coronary arterydisease, oedema, thromboembolism, dysmenorrhea, endometriosis,pre-eclampsia, lung disease, COPD, asthma bone disease, renal disease,glomerulonephritis, liver disease, Crohn's disease, gastritis,ulcerative colitis, ulcer, immune disorder, autoimmune disease,arthritis, rheumatoid arthritis, psoriasis, epidermolysis bullosa,systemic lupus erythematosus, ankylosing spondylitis, Lyme disease,multiple sclerosis, neurodegeneration, stroke, brain/spinal cord injury,Alzheimer's disease, Parkinson's disease, motor neurone disease,neuromuscular disease, HIV, AIDS, cytomegalovirus infection, fungalinfection, ocular disorder, macular degeneration, glaucoma, diabeticretinopathy, ocular hypertension and other conditions in whichimmunoglobulin domain containing cell recognition molecules areimplicated.

[0076] It is particularly preferred that the moieties of the first,second, third, fourth, fifth and sixth aspects of the invention are usedin the manufacture of a medicament for the treatment of an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

[0077] In a thirteenth aspect, the invention provides a method oftreating a disease in a patient comprising administering to the patienta polypeptide of the first aspect of the invention, or a nucleic acidmolecule of the second or third aspect of the invention, or a vector ofthe fourth aspect of the invention, or a host cell of the fifth aspectof the invention, or a ligand of the sixth aspect of the invention, or acompound of the seventh aspect of the invention.

[0078] For diseases in which the expression of a natural gene encoding apolypeptide of the first aspect of the invention, or in which theactivity of a polypeptide of the first aspect of the invention, is lowerin a diseased patient when compared to the level of expression oractivity in a healthy patient, the polypeptide, nucleic acid molecule,ligand or compound administered to the patient should be an agonist.Conversely, for diseases in which the expression of the natural gene oractivity of the polypeptide is higher in a diseased patient whencompared to the level of expression or activity in a healthy patient,the polypeptide, nucleic acid molecule, ligand or compound administeredto the patient should be an antagonist. Examples of such antagonistsinclude antisense nucleic acid molecules, ribozymes and ligands, such asantibodies.

[0079] Preferably, the disease is a disease in which immunoglobulindomain-containing cell surface recognition molecules are implicated, asdescribed above.

[0080] It is particularly preferred that the disease is an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

[0081] In a fourteenth aspect, the invention provides transgenic orknockout non-human animals that have been transformed to express higher,lower or absent levels of a polypeptide of the first aspect of theinvention. Such transgenic animals are very useful models for the studyof disease and may also be used in screening regimes for theidentification of compounds that are effective in the treatment ordiagnosis of such a disease.

[0082] Preferably, the disease is a disease in which immunoglobulindomain-containing cell surface recognition molecules are implicated, asdescribed above.

[0083] It is particularly preferred that the disease is an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

[0084] It should be appreciated that the scope of protection sought forthe polypeptides and nucleic acids of the present invention does notextend to nucleic acids or polypeptides present in their natural source.Rather, the polypeptides and nucleic acids claimed by the presentinvention may be regarded as being “isolated” or “purified”. The terms“isolated” and “purified” as used herein refer to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. Thus, for example, a polypeptide contained in a tissueextract would constitute an “isolated” or “purified” polypeptide, aswould a polypeptide synthetically or recombinantly produced. In oneembodiment, the nucleic acid or polypeptide is found in the presence of(if anything) only a solvent, buffer, ion, or other component normallypresent in a solution of the same.

[0085] It should be noted that the terms “isolated” and “purified” donot denote the method by which the polypeptide or nucleic acid isobtained or the level of purity of the preparation. Thus, such isolatedor purified species may be produced recombinantly, isolated directlyfrom the cell or tissue of interest or produced synthetically based onthe determined sequences.

[0086] A summary of standard techniques and procedures which may beemployed in order to utilise the invention is given below. It will beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, vectors and reagents described. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and it is not intendedthat this terminology should limit the scope of the present invention.The extent of the invention is limited only by the terms of the appendedclaims.

[0087] Standard abbreviations for nucleotides and amino acids are usedin this specification.

[0088] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA technology and immunology, which arewithin the skill of those working in the art.

[0089] Such techniques are explained fully in the literature. Examplesof particularly suitable texts for consultation include the following:Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989);DNA Cloning, Volumes I and II (D. N Glover ed: 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Immunochemical Methods in Cell andMolecular Biology (Mayer and Walker, eds. 1987, Academic Press, London);Scopes, (1987) Protein Purification: Principles and Practice, SecondEdition (Springer Verlag, N.Y.); and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds. 1986).

[0090] As used herein, the term “polypeptide” includes any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e. peptide isosteres. Thisterm refers both to short chains (peptides and oligopeptides) and tolonger chains (proteins).

[0091] The polypeptide of the present invention may be in the form of amature protein or may be a pre-, pro- or prepro-protein that can beactivated by cleavage of the pre-, pro- or prepro-portion to produce anactive mature polypeptide. In such polypeptides, the pre-, pro- orprepro-sequence may be a leader or secretory sequence or may be asequence that is employed for purification of the mature polypeptidesequence.

[0092] The polypeptide of the first aspect of the invention may formpart of a fusion protein. For example, it is often advantageous toinclude one or more additional amino acid sequences which may containsecretory or leader sequences, pro-sequences, sequences which aid inpurification, or sequences that confer higher protein stability, forexample during recombinant production. Alternatively or additionally,the mature polypeptide may be fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol).

[0093] Polypeptides may contain amino acids other than the 20gene-encoded amino acids, modified either by natural processes, such asby post-translational processing or by chemical modification techniqueswhich are well known in the art. Among the known modifications which maycommonly be present in polypeptides of the present invention areglycosylation, lipid attachment, sulphation, gamma-carboxylation, forinstance of glutamic acid residues, hydroxylation and ADP-ribosylation.Other potential modifications include acetylation, acylation, amidation,covalent attachment of flavin, covalent attachment of a haeme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulphide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation, GPI anchorformation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

[0094] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl terminus in apolypeptide, or both, by a covalent modification is common innaturally-occurring and synthetic polypeptides and such modificationsmay be present in polypeptides of the present invention.

[0095] The modifications that occur in a polypeptide often will be afunction of how the polypeptide is made. For polypeptides that are maderecombinantly, the nature and extent of the modifications in large partwill be determined by the post-translational modification capacity ofthe particular host cell and the modification signals that are presentin the amino acid sequence of the polypeptide in question. For instance,glycosylation patterns vary between different types of host cell.

[0096] The polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally-occurringpolypeptides (for example purified from cell culture),recombinantly-produced polypeptides (including fusion proteins),synthetically-produced polypeptides or polypeptides that are produced bya combination of these methods.

[0097] The functionally-equivalent polypeptides of the first aspect ofthe invention may be polypeptides that are homologous to the INSP052 andINSP055 polypeptides, preferably the INSP052 extracellular domain (i.e.SEQ ID NO:20 or SEQ ID NO:22). Two polypeptides are said to be“homologous”, as the term is used herein, if the sequence of one of thepolypeptides has a high enough degree of identity or similarity to thesequence of the other polypeptide. “Identity” indicates that at anyparticular position in the aligned sequences, the amino acid residue isidentical between the sequences. “Similarity” indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. Degrees of identity andsimilarity can be readily calculated (Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing.Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991).

[0098] Additionally, homology comparisons can be conducted by eye, ormore usually, with the aid of readily available sequence comparisonprograms. These commercially available computer programs can calculate %homology between two or more sequences.

[0099] % homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

[0100] Although this is a very simple and consistent method, it fails totake into consideration that, for example, in an otherwise identicalpair of sequences, one insertion or deletion will cause the followingamino acid residues to be put out of alignment, thus potentiallyresulting in a large reduction in % homology when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without penalising unduly the overall homologyscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximise local homology.

[0101] However, these more complex methods assign “gap penalties” toeach gap that occurs in the alignment so that, for the same number ofidentical amino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and -4 for each extension.

[0102] Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

[0103] Although the final % homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or -nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). It is preferred to usethe public default values for the GCG package, or in the case of othersoftware, the default matrix, such as BLOSUM62.

[0104] Once the software has produced an optimal alignment, it ispossible to calculate % homology, preferably % sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

[0105] The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence.

[0106] Homologous polypeptides therefore include natural biologicalvariants (for example, allelic variants or geographical variationswithin the species from which the polypeptides are derived) and mutants(such as mutants containing amino acid substitutions, insertions ordeletions) of the INSP052 and INSP055 polypeptides, preferably of theINSP052 extracellular domain. Such mutants may include polypeptides inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; among the basic residues Lysand Arg; or among the aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, i.e. between 5 and 10, 1 and 5,1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or addedin any combination. Especially preferred are silent substitutions,additions and deletions, which do not alter the properties andactivities of the protein. Also especially preferred in this regard areconservative substitutions. Such mutants also include polypeptides inwhich one or more of the amino acid residues includes a substituentgroup.

[0107] Typically, greater than 30% identity between two polypeptides isconsidered to be an indication of functional equivalence. Preferably,functionally equivalent polypeptides of the first aspect of theinvention have a degree of sequence identity with the INSP052 andINSP055 polypeptides, or with active fragments thereof, of greater than80%. More preferred polypeptides have degrees of identity of greaterthan 85%, 90%, 95%, 98% or 99%, respectively.

[0108] The functionally-equivalent polypeptides of the first aspect ofthe invention may also be polypeptides which have been identified usingone or more techniques of structural alignment. For example, theInpharmatica Genome Threader™ technology that forms one aspect of thesearch tools used to generate the Biopendium search database may be used(see co-pending International Patent Application No. PCT/GB01/01105,published as WO 01/69507) to identify polypeptides of presently-unknownfunction which, while having low sequence identity as compared to theINSP052 and INSP055 polypeptides, are predicted to be immunoglobulindomain-containing cell surface recognition molecules, said methodutilising a polypeptide of the first aspect of the invention, by virtueof sharing significant structural homology with the INSP052 and INSP055polypeptide sequences. By “significant structural homology” is meantthat the Inpharmatica Genome Threader™ predicts two proteins to sharestructural homology with a certainty of at least 10% and morepreferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.

[0109] The polypeptides of the first aspect of the invention alsoinclude fragments of the INSP052 and INSP055 polypeptides and fragmentsof the functional equivalents of the INSP052 and INSP055 polypeptides,provided that those fragments retain immunoglobulin domain-containingcell surface recognition molecule activity or have an antigenicdeterminant in common with the INSP052 and INSP055 polypeptides.

[0110] As used herein, the term “fragment” refers to a polypeptidehaving an amino acid sequence that is the same as part, but not all, ofthe amino acid sequence of the INSP052 and INSP055 polypeptides or oneof its functional equivalents. The fragments should comprise at least nconsecutive amino acids from the sequence and, depending on theparticular sequence, n preferably is 7 or more (for example, 8, 10, 12,14, 16, 18, 20 or more). Small fragments may form an antigenicdeterminant.

[0111] Such fragments may be “free-standing”, i.e. not part of or fusedto other amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the fragment of the invention mostpreferably forms a single continuous region. For instance, certainpreferred embodiments relate to a fragment having a pre- and/orpro-polypeptide region fused to the amino terminus of the fragmentand/or an additional region fused to the carboxyl terminus of thefragment. However, several fragments may be comprised within a singlelarger polypeptide.

[0112] The polypeptides of the present invention or their immunogenicfragments (comprising at least one antigenic determinant) can be used togenerate ligands, such as polyclonal or monoclonal antibodies, that areimmunospecific for the polypeptides. Such antibodies may be employed toisolate or to identify clones expressing the polypeptides of theinvention or to purify the polypeptides by affinity chromatography. Theantibodies may also be employed as diagnostic or therapeutic aids,amongst other applications, as will be apparent to the skilled reader.

[0113] The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art. Asused herein, the term “antibody” refers to intact molecules as well asto fragments thereof, such as Fab, F(ab′)₂ and Fv, which are capable ofbinding to the antigenic determinant in question. Such antibodies thusbind to the polypeptides of the first aspect of the invention.

[0114] By “substantially greater affinity” we mean that there is ameasurable increase in the affinity for a polypeptide of the inventionas compared with the affinity for known immunoglobluin domain-containingcell surface recognition molecules.

[0115] Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold10-fold, 100-fold, 10³-fold, 10⁴-fold, 10⁵-fold or 10⁶-fold greater fora polypeptide of the invention than for known immunoglobluindomain-containing cell surface recognition molecules.

[0116] If polyclonal antibodies are desired, a selected mammal, such asa mouse, rabbit, goat or horse, may be immunised with a polypeptide ofthe first aspect of the invention. The polypeptide used to immunise theanimal can be derived by recombinant DNA technology or can besynthesized chemically. If desired, the polypeptide can be conjugated toa carrier protein. Commonly used carriers to which the polypeptides maybe chemically coupled include bovine serum albumin, thyroglobulin andkeyhole limpet haemocyanin. The coupled polypeptide is then used toimmunise the animal. Serum from the immunised animal is collected andtreated according to known procedures, for example by immunoaffinitychromatography.

[0117] Monoclonal antibodies to the polypeptides of the first aspect ofthe invention can also be readily produced by one skilled in the art.The general methodology for making monoclonal antibodies using hybridomatechnology is well known (see, for example, Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72(1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985).

[0118] Panels of monoclonal antibodies produced against the polypeptidesof the first aspect of the invention can be screened for variousproperties, i.e., for isotype, epitope, affinity, etc. Monoclonalantibodies are particularly useful in purification of the individualpolypeptides against which they are directed. Alternatively, genesencoding the monoclonal antibodies of interest may be isolated fromhybridomas, for instance by PCR techniques known in the art, and clonedand expressed in appropriate vectors.

[0119] Chimeric antibodies, in which non-human variable regions arejoined or fused to human constant regions (see, for example, Liu et al.,Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.

[0120] The antibody may be modified to make it less immunogenic in anindividual, for example by humanisation (see Jones et al., Nature, 321,522 (1986); Verhoeyen et al., Science, 239, 1534 (1988); Kabat et al.,J. Immunol., 147, 1709 (1991); Queen et al., Proc. Natl. Acad. Sci. USA,86, 10029 (1989); Gorman et al., Proc. Natl. Acad. Sci. USA, 88, 34181(1991); and Hodgson et al., Bio/Technology, 9, 421 (1991)). The term“humanised antibody”, as used herein, refers to antibody molecules inwhich the CDR amino acids and selected other amino acids in the variabledomains of the heavy and/or light chains of a non-human donor antibodyhave been substituted in place of the equivalent amino acids in a humanantibody. The humanised antibody thus closely resembles a human antibodybut has the binding ability of the donor antibody.

[0121] In a further alternative, the antibody may be a “bispecific”antibody, that is an antibody having two different antigen bindingdomains, each domain being directed against a different epitope.

[0122] Phage display technology may be utilised to select genes whichencode antibodies with binding activities towards the polypeptides ofthe invention either from repertoires of PCR amplified V-genes oflymphocytes from humans screened for possessing the relevant antibodies,or from naive libraries (McCafferty, J. et al., (1990), Nature 348,552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). Theaffinity of these antibodies can also be improved by chain shuffling(Clackson, T. et al., (1991) Nature 352, 624-628).

[0123] Antibodies generated by the above techniques, whether polyclonalor monoclonal, have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme.

[0124] Preferred nucleic acid molecules of the second and third aspectsof the invention are those which encode the polypeptide sequencesrecited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14 and/or SEQ ID NO:16, SEQ ID NO:18, theextracellular domain of INSP052 (SEQ ID NO:20 and SEQ ID NO:22), SEQ IDNO:24, or SEQ ID NO:26 and functionally equivalent polypeptides, e.g.fusion proteins consisting of the extracellular domain of INSP052 fusedto one or more additional polypeptide sequences. These nucleic acidmolecules may be used in the methods and applications described herein.The nucleic acid molecules of the invention preferably comprise at leastn consecutive nucleotides from the sequences disclosed herein where,depending on the particular sequence, n is 10 or more (for example, 12,14, 15, 18, 20, 25, 30, 35, 40 or more).

[0125] The nucleic acid molecules of the invention also includesequences that are complementary to nucleic acid molecules describedabove (for example, for antisense or probing purposes).

[0126] Nucleic acid molecules of the present invention may be in theform of RNA, such as mRNA, or in the form of DNA, including, forinstance cDNA, synthetic DNA or genomic DNA. Such nucleic acid moleculesmay be obtained by cloning, by chemical synthetic techniques or by acombination thereof. The nucleic acid molecules can be prepared, forexample, by chemical synthesis using techniques such as solid phasephosphoramidite chemical synthesis, from genomic or cDNA libraries or byseparation from an organism. RNA molecules may generally be generated bythe in vitro or in vivo transcription of DNA sequences.

[0127] The nucleic acid molecules may be double-stranded orsingle-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

[0128] The term “nucleic acid molecule” also includes analogues of DNAand RNA, such as those containing modified backbones, and peptidenucleic acids (PNA). The term “PNA”, as used herein, refers to anantisense molecule or an anti-gene agent which comprises anoligonucleotide of at least five nucleotides in length linked to apeptide backbone of amino acid residues, which preferably ends inlysine. The terminal lysine confers solubility to the composition. PNAsmay be pegylated to extend their lifespan in a cell, where theypreferentially bind complementary single stranded DNA and RNA and stoptranscript elongation (Nielsen, P. E. et al. (1993) Anticancer Drug Des.8:53-63).

[0129] These molecules also may have a different sequence which, as aresult of the degeneracy of the genetic code, encode a polypeptide ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14 and/or SEQ ID NO:16, or SEQ ID NO:18, or theextracellular domain of INSP052 or SEQ ID NO:24 or SEQ ID NO:26. Suchmolecules may include, but are not limited to, the coding sequence forthe mature polypeptide by itself; the coding sequence for the maturepolypeptide and additional coding sequences, such as those encoding aleader or secretory sequence, such as a pro-, pre- or prepro-polypeptidesequence; the coding sequence of the mature polypeptide, with or withoutthe aforementioned additional coding sequences, together with furtheradditional, non-coding sequences, including non-coding 5′ and 3′sequences, such as the transcribed, non-translated sequences that play arole in transcription (including termination signals), ribosome bindingand mRNA stability. The nucleic acid molecules may also includeadditional sequences which encode additional amino acids, such as thosewhich provide additional functionalities.

[0130] The nucleic acid molecules of the second and third aspects of theinvention may also encode the fragments or the functional equivalents ofthe polypeptides and fragments of the first aspect of the invention.Such a nucleic acid molecule may be a naturally-occurring variant suchas a naturally-occurring allelic variant, or the molecule may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the nucleic acid molecule may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells or organisms.

[0131] Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or insertions. The substitutions, deletions or insertions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or insertions.

[0132] The nucleic acid molecules of the invention can also beengineered, using methods generally known in the art, for a variety ofreasons, including modifying the cloning, processing, and/or expressionof the gene product (the polypeptide). DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides are included as techniques which may be used toengineer the nucleotide sequences. Site-directed mutagenesis may be usedto insert new restriction sites, alter glycosylation patterns, changecodon preference, produce splice variants, introduce mutations and soforth.

[0133] Nucleic acid molecules which encode a polypeptide of the firstaspect of the invention may be ligated to a heterologous sequence sothat the combined nucleic acid molecule encodes a fusion protein. Suchcombined nucleic acid molecules are included within the second or thirdaspects of the invention. For example, to screen peptide libraries forinhibitors of the activity of the polypeptide, it may be useful toexpress, using such a combined nucleic acid molecule, a fusion proteinthat can be recognised by a commercially-available antibody. A fusionprotein may also be engineered to contain a cleavage site locatedbetween the sequence of the polypeptide of the invention and thesequence of a heterologous protein so that the polypeptide may becleaved and purified away from the heterologous protein.

[0134] The nucleic acid molecules of the invention also includeantisense molecules that are partially complementary to nucleic acidmolecules encoding polypeptides of the present invention and thattherefore hybridize to the encoding nucleic acid molecules(hybridization). Such antisense molecules, such as oligonucleotides, canbe designed to recognise, specifically bind to and prevent transcriptionof a target nucleic acid encoding a polypeptide of the invention, aswill be known by those of ordinary skill in the art (see, for example,Cohen, J. S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J.Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee etal., Nucleic Acids Res 6, 3073 (1979); Cooney et al., Science 241, 456(1988); Dervan et al., Science 251, 1360 (1991).

[0135] The term “hybridization” as used herein refers to the associationof two nucleic acid molecules with one another by hydrogen bonding.Typically, one molecule will be fixed to a solid support and the otherwill be free in solution. Then, the two molecules may be placed incontact with one another under conditions that favour hydrogen bonding.Factors that affect this bonding include: the type and volume ofsolvent; reaction temperature; time of hybridization; agitation; agentsto block the non-specific attachment of the liquid phase molecule to thesolid support (Denhardt's reagent or BLOTTO); the concentration of themolecules; use of compounds to increase the rate of association ofmolecules (dextran sulphate or polyethylene glycol); and the stringencyof the washing conditions following hybridization (see Sambrook et al.[supra]).

[0136] The inhibition of hybridization of a completely complementarymolecule to a target molecule may be examined using a hybridizationassay, as known in the art (see, for example, Sambrook et al [supra]). Asubstantially homologous molecule will then compete for and inhibit thebinding of a completely homologous molecule to the target molecule undervarious conditions of stringency, as taught in Wahl, G. M. and S. L.Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

[0137] “Stringency” refers to conditions in a hybridization reactionthat favour the association of very similar molecules over associationof molecules that differ. High stringency hybridisation conditions aredefined as overnight incubation at 42° C. in a solution comprising 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5× Denhardts solution, 10% dextran sulphate, and 20microgram/ml denatured, sheared salmon sperm DNA, followed b y washingthe filters in 0.1×SSC at approximately 65° C. Low stringency conditionsinvolve the hybridisation reaction being carried out at 35° C. (seeSambrook et al. [supra]). Preferably, the conditions used forhybridization are those of high stringency.

[0138] Preferred embodiments of this aspect of the invention are nucleicacid molecules that are at least 70% identical over their entire lengthto a nucleic acid molecule encoding the INSP052 or INSP055 polypeptides(SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13 and/or SEQ ID NO:15, or SEQ ID NO:17, or the nucleicacid sequence set forth in FIG. 7 or the coding portion of the nucleicacid sequence set forth in FIG. 7 (i.e. SEQ ID NO: 19 or SEQ ID NO: 21),SEQ ID NO: 23, or SEQ ID NO: 25) and nucleic acid molecules that aresubstantially complementary to such nucleic acid molecules. Preferably,a nucleic acid molecule according to this aspect of the inventioncomprises a region that is at least 80% identical over its entire lengthto the coding sequence for SEQ ID NO:2 given in SEQ ID NO:1, the codingsequence for SEQ ID NO:4 given in SEQ ID NO:3, the coding sequences forSEQ ID NO:6 given in SEQ ID NO:5, the coding sequence for SEQ ID NO:8given in SEQ ID NO:7, the coding sequence for SEQ ID NO:10 given in SEQID NO:9, the coding sequence for SEQ ID NO:12 given in SEQ ID NO:11, thecoding sequence for SEQ ID NO:14 given in SEQ ID NO:13, the codingsequence for SEQ ID NO:16 given in SEQ ID NO:15, the coding sequence forSEQ ID NO:18 given in SEQ ID NO:17, the coding sequence for SEQ ID NO:24given in SEQ ID NO:23, the coding sequence for SEQ ID NO: 26 given inSEQ ID NO: 25, or is a nucleic acid molecule that is complementarythereto. Particularly preferred is a nucleic acid which comprises orconsists of a region that is at least 80% identical over its entirelength to the coding sequence for the extracellular domain of INSP052(the extracellular domain of mature INSP052 or the extracellular domainof INSP052 comprising the signal peptide) as given in FIG. 7. In thisregard, nucleic acid molecules at least 90%, preferably at least 95%,more preferably at least 98% or 99% identical over their entire lengthto the same are particularly preferred. Preferred embodiments in thisrespect are nucleic acid molecules that encode polypeptides which retainsubstantially the same biological function or activity as the INSP052and INSP055 polypeptides.

[0139] The invention also provides a process for detecting a nucleicacid molecule of the invention, comprising the steps of: (a) contactinga nucleic probe according to the invention with a biological sampleunder hybridizing conditions to form duplexes; and (b) detecting anysuch duplexes that are formed.

[0140] As discussed additionally below in connection with assays thatmay be utilised according to the invention, a nucleic acid molecule asdescribed above may be used as a hybridization probe for RNA, cDNA orgenomic DNA, in order to isolate full-length cDNAs and genomic clonesencoding the INSP052 and INSP055 polypeptides and to isolate cDNA andgenomic clones of homologous or orthologous genes that have a highsequence similarity to the gene encoding this polypeptide.

[0141] In this regard, the following techniques, among others known inthe art, may be utilised and are discussed below for purposes ofillustration. Methods for DNA sequencing and analysis are well known andare generally available in the art and may, indeed, be used to practicemany of the embodiments of the invention discussed herein. Such methodsmay employ such enzymes as the Klenow fragment of DNA polymerase I,Sequenase (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (PerkinElmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of polymerases and proof-reading exonucleases such as thosefound in the ELONGASE Amplification System marketed by Gibco/BRL(Gaithersburg, Md.). Preferably, the sequencing process may be automatedusing machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

[0142] One method for isolating a nucleic acid molecule encoding apolypeptide with an equivalent function to that of the INSP052 andINSP055 polypeptides is to probe a genomic or cDNA library with anatural or artificially-designed probe using standard procedures thatare recognised in the art (see, for example, “Current Protocols inMolecular Biology”, Ausubel et al. (eds). Greene Publishing Associationand John Wiley Interscience, New York, 1989,1992). Probes comprising atleast 15, preferably at least 30, and more preferably at least 50,contiguous bases that correspond to, or are complementary to, nucleicacid sequences from the appropriate encoding gene (SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25) are particularly useful probes. Such probes may belabelled with an analytically-detectable reagent to facilitate theiridentification. Useful reagents include, but are not limited to,radioisotopes, fluorescent dyes and enzymes that are capable ofcatalysing the formation of a detectable product. Using these probes,the ordinarily skilled artisan will be capable of isolatingcomplementary copies of genomic DNA, cDNA or RNA polynucleotidesencoding proteins of interest from human, mammalian or other animalsources and screening such sources for related sequences, for example,for additional members of the family, type and/or subtype.

[0143] In many cases, isolated cDNA sequences will be incomplete, inthat the region encoding the polypeptide will be cut short, normally atthe 5′ end. Several methods are available to obtain full length cDNAs,or to extend short cDNAs. Such sequences may be extended utilising apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed is based on themethod of Rapid Amplification of cDNA Ends (RACE; see, for example,Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent modifications ofthis technique, exemplified by the Marathon™ technology (ClontechLaboratories Inc.), for example, have significantly simplified thesearch for longer cDNAs. A slightly different technique, termed“restriction-site” PCR, uses universal primers to retrieve unknownnucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCRMethods Applic. 2:318-322). Inverse PCR may also be used to amplify orto extend sequences using divergent primers based on a known region(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic., 1,111-119). Another method which may be used to retrieve unknown sequencesis that of Parker, J. D. et al. (1991); Nucleic Acids Res.19:3055-3060). Additionally, one may use PCR, nested primers, andPromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

[0144] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

[0145] In one embodiment of the invention, the nucleic acid molecules ofthe present invention may be used for chromosome localisation. In thistechnique, a nucleic acid molecule is specifically targeted to, and canhybridize with, a particular location on an individual human chromosome.The mapping of relevant sequences to chromosomes according to thepresent invention is an important step in the confirmatory correlationof those sequences with the gene-associated disease. Once a sequence hasbeen mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data.Such data are found in, for example, V. McKusick, Mendelian Inheritancein Man (available on-line through Johns Hopkins University Welch MedicalLibrary). The relationships between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localised by genetic linkage toa particular genomic region, any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleic acid molecule may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier, or affected individuals.

[0146] The nucleic acid molecules of the present invention are alsovaluable for tissue localisation such techniques allow the determinationof expression patterns of the polypeptide in tissues by detection of themRNAs that encode them. These techniques include in situ hybridizationtechniques and nucleotide amplification techniques, such as PCR. Resultsfrom these studies provide an indication of the normal functions of thepolypeptide in the organism. In addition, comparative studies of thenormal expression pattern of mRNAs with that of mRNAs encoded by amutant gene provide valuable insights into the role of mutantpolypeptides in disease. Such inappropriate expression may be of atemporal, spatial or quantitative nature.

[0147] Gene silencing approaches may also be undertaken to down-regulateendogenous expression of a gene encoding a polypeptide of the invention.RNA interference (RNAi) (Elbashir, S M et al., Nature 2001, 411,494-498) is one method of sequence specific post-transcriptional genesilencing that may be employed. Short dsRNA oligonucleotides aresynthesised in vitro and introduced into a cell. The sequence specificbinding of these dsRNA oligonucleotides triggers the degradation oftarget mRNA, reducing or ablating target protein expression.

[0148] Efficacy of the gene silencing approaches assessed above may beassessed through the measurement of polypeptide expression (for example,by Western blotting), and at the RNA level using TaqMan-basedmethodologies.

[0149] The vectors of the present invention comprise nucleic acidmolecules of the invention and may be cloning or expression vectors. Thehost cells of the invention, which may be transformed, transfected ortransduced with the vectors of the invention may be prokaryotic oreukaryotic.

[0150] The polypeptides of the invention may be prepared in recombinantform by expression of their encoding nucleic acid molecules in vectorscontained within a host cell. Such expression methods are well known tothose of skill in the art and many are described in detail by Sambrooket al (supra) and Fernandez & Hoeffler (1998, eds. “Gene expressionsystems. Using nature for the art of expression”. Academic Press, SanDiego, London, Boston, New York, Sydney, Tokyo, Toronto).

[0151] Generally, any system or vector that is suitable to maintain,propagate or express nucleic acid molecules to produce a polypeptide inthe required host may be used. The appropriate nucleotide sequence maybe inserted into an expression system by any of a variety of well-knownand routine techniques, such as, for example, those described inSambrook et al., (supra). Generally, the encoding gene can be placedunder the control of a control element such as a promoter, ribosomebinding site (for bacterial expression) and, optionally an operator, sothat the DNA sequence encoding the desired polypeptide is transcribedinto RNA in the transformed host cell.

[0152] Examples of suitable expression systems include, for example,chromosomal, episomal and virus-derived systems, including, for example,vectors derived from: bacterial plasmids, bacteriophage, transposons,yeast episomes, insertion elements, yeast chromosomal elements, virusessuch as baculoviruses, papova viruses such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,or combinations thereof, such as those derived from plasmid andbacteriophage genetic elements, including cosmids and phagemids. Humanartificial chromosomes (HACs) may also be employed to deliver largerfragments of DNA than can be contained and expressed in a plasmid.

[0153] Particularly suitable expression systems include microorganismssuch as bacteria transformed with recombinant bacteriophage, plasmid orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors (forexample, baculovirus); plant cell systems transformed with virusexpression vectors (for example, cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or with bacterial expression vectors (for example, Tior pBR322 plasmids); or animal cell systems.

[0154] Cell-free translation systems can also be employed to produce thepolypeptides of the invention.

[0155] Introduction of nucleic acid molecules encoding a polypeptide ofthe present invention into host cells can be effected by methodsdescribed in many standard laboratory manuals, such as Davis et al.,Basic Methods in Molecular Biology (1986) and Sambrook et al., [supra].Particularly suitable methods include calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection (see Sambrook etal., 1989 [supra]; Ausubel et al., 1991 [supra]; Spector, Goldman &Leinwald, 1998). In eukaryotic cells, expression systems may either betransient (for example, episomal) or permanent (chromosomal integration)according to the needs of the system.

[0156] The encoding nucleic acid molecule may or may not include asequence encoding a control sequence, such as a signal peptide or leadersequence, as desired, for example, for secretion of the translatedpolypeptide into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment. These signalsmay be endogenous to the polypeptide or they may be heterologoussignals. Leader sequences can be removed by the bacterial host inpost-translational processing.

[0157] In addition to control sequences, it may be desirable to addregulatory sequences that allow for regulation of the expression of thepolypeptide relative to the growth of the host cell. Examples ofregulatory sequences are those which cause the expression of a gene tobe increased or decreased in response to a chemical or physicalstimulus, including the presence of a regulatory compound or to varioustemperature or metabolic conditions. Regulatory sequences are thosenon-translated regions of the vector, such as enhancers, promoters and5′ and 3′ untranslated regions. These interact with host cellularproteins to carry out transcription and translation. Such regulatorysequences may vary in their strength and specificity. Depending on thevector system and host utilised, any number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, may be used. For example, when cloning in bacterial systems,inducible promoters such as the hybrid lacZ promoter of the Bluescriptphagemid (Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL)and the like may be used. The baculovirus polyhedrin promoter may beused in insect cells. Promoters or enhancers derived from the genomes ofplant cells (for example, heat shock, RUBISCO and storage protein genes)or from plant viruses (for example, viral promoters or leader sequences)may be cloned into the vector. In mammalian cell systems, promoters frommammalian genes or from mammalian viruses are preferable. If it isnecessary to generate a cell line that contains multiple copies of thesequence, vectors based on SV40 or EBV may be used with an appropriateselectable marker.

[0158] An expression vector is constructed so that the particularnucleic acid coding sequence is located in the vector with theappropriate regulatory sequences, the positioning and orientation of thecoding sequence with respect to the regulatory sequences being such thatthe coding sequence is transcribed under the “control” of the regulatorysequences, i.e., RNA polymerase which binds to the DNA molecule at thecontrol sequences transcribes the coding sequence. In some cases it maybe necessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; i.e., to maintainthe reading frame.

[0159] The control sequences and other regulatory sequences may beligated to the nucleic acid coding sequence prior to insertion into avector. Alternatively, the coding sequence can be cloned directly intoan expression vector that already contains the control sequences and anappropriate restriction site.

[0160] For long-term, high-yield production of a recombinantpolypeptide, stable expression is preferred. For example, cell lineswhich stably express the polypeptide of interest may be transformedusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells that successfully express theintroduced sequences. Resistant clones of stably transformed cells maybe proliferated using tissue culture techniques appropriate to the celltype.

[0161] Mammalian cell lines available as hosts for expression are knownin the art and include many immortalised cell lines available from theAmerican Type Culture Collection (ATCC) including, but not limited to,Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkeykidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and humanhepatocellular carcinoma (for example Hep G2) cells and a number ofother cell lines.

[0162] In the baculovirus system, the materials for baculovirus/insectcell expression systems are commercially available in kit form from,inter alia, Invitrogen, San Diego Calif. (the “MaxBac” kit). Thesetechniques are generally known to those skilled in the art and aredescribed fully in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987). Particularly suitable host cells foruse in this system include insect cells such as Drosophila S2 andSpodoptera Sf9 cells.

[0163] There are many plant cell culture and whole plant geneticexpression systems known in the art. Examples of suitable plant cellulargenetic expression systems include those described in U.S. Pat. No.5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143.Additional examples of genetic expression in plant cell culture has beendescribed by Zenk, Phytochemistry 30, 3861-3863 (1991).

[0164] In particular, all plants from which protoplasts can be isolatedand cultured to give whole regenerated plants can be utilised, so thatwhole plants are recovered which contain the transferred gene.Practically all plants can be regenerated from cultured cells ortissues, including but not limited to all major species of sugar cane,sugar beet, cotton, fruit and other trees, legumes and vegetables.

[0165] Examples of particularly preferred bacterial host cells includestreptococci, staphylococci, E. coli, Streptomyces and Bacillus subtiliscells.

[0166] Examples of particularly suitable host cells for fungalexpression include yeast cells (for example, S. cerevisiae) andAspergillus cells.

[0167] Any number of selection systems are known in the art that may beused to recover transformed cell lines. Examples include the herpessimplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32)and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell22:817-23) genes that can be employed in tk- or aprt± cells,respectively.

[0168] Also, antimetabolite, antibiotic or herbicide resistance can beused as the basis for selection; for example, dihydrofolate reductase(DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively.Additional selectable genes have been described, examples of which willbe clear to those of skill in the art.

[0169] Although the presence or absence of marker gene expressionsuggests that the gene of interest is also present, its presence andexpression may need to be confirmed. For example, if the relevantsequence is inserted within a marker gene sequence, transformed cellscontaining the appropriate sequences can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding a polypeptide of the invention under thecontrol of a single promoter. Expression of the marker gene in responseto induction or selection usually indicates expression of the tandemgene as well.

[0170] Alternatively, host cells that contain a nucleic acid sequenceencoding a polypeptide of the invention and which express saidpolypeptide may be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example,fluorescence activated cell sorting (FACS) or immunoassay techniques(such as the enzyme-linked immunosorbent assay [ELISA] andradioimmunoassay [RIA]), that include membrane, solution, or chip basedtechnologies for the detection and/or quantification of nucleic acid orprotein (see Hampton, R. et al. (1990) Serological Methods, a LaboratoryManual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983) J.Exp. Med, 158, 1211-1216).

[0171] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labelled hybridization or PCRprobes for detecting sequences related to nucleic acid moleculesencoding polypeptides of the present invention include oligolabelling,nick translation, end-labelling or PCR amplification using a labelledpolynucleotide. Alternatively, the sequences encoding the polypeptide ofthe invention may be cloned into a vector for the production of an mRNAprobe. Such vectors are known in the art, are commercially available,and may be used to synthesise RNA probes in vitro by addition of anappropriate RNA polymerase such as T7, T3 or SP6 and labellednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)).

[0172] Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes and fluorescent,chemiluminescent or chromogenic agents as well as substrates, cofactors,inhibitors, magnetic particles, and the like.

[0173] Nucleic acid molecules according to the present invention mayalso be used to create transgenic animals, particularly rodent animals.Such transgenic animals form a further aspect of the present invention.This may be done locally by modification of somatic cells, or by germline therapy to incorporate heritable modifications. Such transgenicanimals may be particularly useful in the generation of animal modelsfor drug molecules effective as modulators of the polypeptides of thepresent invention.

[0174] The polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulphate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. High performance liquid chromatography isparticularly useful for purification. Well known techniques forrefolding proteins may be employed to regenerate an active conformationwhen the polypeptide is denatured during isolation and or purification.

[0175] Specialised vector constructions may also be used to facilitatepurification of proteins, as desired, by joining sequences encoding thepolypeptides of the invention to a nucleotide sequence encoding apolypeptide domain that will facilitate purification of solubleproteins.

[0176] Examples of such purification-facilitating domains include metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilised inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the polypeptide of theinvention may be used to facilitate purification. One such expressionvector provides for expression of a fusion protein containing thepolypeptide of the invention fused to several histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification by IMAC (immobilised metal ion affinitychromatography as described in Porath, J. et al. (1992), Prot. Exp.Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage siteprovides a means for purifying the polypeptide from the fusion protein.A discussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0177] If the polypeptide is to be expressed for use in screeningassays, generally it is preferred that it be produced at the surface ofthe host cell in which it is expressed. In this event, the host cellsmay be harvested prior to use in the screening assay, for example usingtechniques such as fluorescence activated cell sorting (FACS) orimmunoaffinity techniques. If the polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify theexpressed polypeptide. If polypeptide is produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0178] The polypeptide of the invention can be used to screen librariesof compounds in any of a variety of drug screening techniques. Suchcompounds may activate (agonise) or inhibit (antagonise) the level ofexpression of the gene or the activity of the polypeptide of theinvention and form a further aspect of the present invention. Preferredcompounds are effective to alter the expression of a natural gene whichencodes a polypeptide of the first aspect of the invention or toregulate the activity of a polypeptide of the first aspect of theinvention.

[0179] Agonist or antagonist compounds may be isolated from, forexample, cells, cell-free preparations, chemical libraries or naturalproduct mixtures. These agonists or antagonists may be natural ormodified substrates, ligands, enzymes, receptors or structural orfunctional mimetics. For a suitable review of such screening techniques,see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991).

[0180] Compounds that are most likely to be good antagonists aremolecules that bind to the polypeptide of the invention without inducingthe biological effects of the polypeptide upon binding to it. Potentialantagonists include small organic molecules, peptides, polypeptides andantibodies that bind to the polypeptide of the invention and therebyinhibit or extinguish its activity. In this fashion, binding of thepolypeptide to normal cellular binding molecules may be inhibited, suchthat the normal biological activity of the polypeptide is prevented.

[0181] The polypeptide of the invention that is employed in such ascreening technique may be free in solution, affixed to a solid support,borne on a cell surface or located intracellularly. In general, suchscreening procedures may involve using appropriate cells or cellmembranes that express the polypeptide that are contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response. The, functional response of the cells contactedwith the test compound is then compared with control cells that were notcontacted with the test compound. Such an assay may assess whether thetest compound results in a signal generated by activation of thepolypeptide, using an appropriate detection system. Inhibitors ofactivation are generally assayed in the presence of a known agonist andthe effect on activation by the agonist in the presence of the testcompound is observed.

[0182] A preferred method for identifying an agonist or antagonistcompound of a polypeptide of the present invention comprises:

[0183] (a) contacting a cell expressing on the surface thereof thepolypeptide according to the first aspect of the invention, thepolypeptide being associated with a second component capable ofproviding a detectable signal in response to the binding of a compoundto the polypeptide, with a compound to be screened under conditions topermit binding to the polypeptide; and

[0184] (b) determining whether the compound binds to and activates orinhibits the polypeptide by measuring the level of a signal generatedfrom the interaction of the compound with the polypeptide.

[0185] A further preferred method for identifying an agonist orantagonist of a polypeptide of the invention comprises:

[0186] (a) contacting a cell expressing on the surface thereof thepolypeptide, the polypeptide being associated with a second componentcapable of providing a detectable signal in response to the binding of acompound to the polypeptide, with a compound to be screened underconditions to permit binding to the polypeptide; and

[0187] (b) determining whether the compound binds to and activates orinhibits the polypeptide by comparing the level of a signal generatedfrom the interaction of the compound with the polypeptide with the levelof a signal in the absence of the compound.

[0188] In further preferred embodiments, the general methods that aredescribed above may further comprise conducting the identification ofagonist or antagonist in the presence of labelled or unlabelled ligandfor the polypeptide.

[0189] In another embodiment of the method for identifying agonist orantagonist of a polypeptide of the present invention comprises:

[0190] determining the inhibition of binding of a ligand to cells whichhave a polypeptide of the invention on the surface thereof, or to cellmembranes containing such a polypeptide, in the presence of a candidatecompound under conditions to permit binding to the polypeptide, anddetermining the amount of ligand bound to the polypeptide. A compoundcapable of causing reduction of binding of a ligand is considered to bean agonist or antagonist. Preferably the ligand is labelled.

[0191] More particularly, a method of screening for a polypeptideantagonist or agonist compound comprises the steps of:

[0192] (a) incubating a labelled ligand with a whole cell expressing apolypeptide according to the invention on the cell surface, or a cellmembrane containing a polypeptide of the invention,

[0193] (b) measuring the amount of labelled ligand bound to the wholecell or the cell membrane;

[0194] (c) adding a candidate compound to a mixture of labelled ligandand the whole cell or the cell membrane of step (a) and allowing themixture to attain equilibrium;

[0195] (d) measuring the amount of labelled ligand bound to the wholecell or the cell membrane after step (c); and

[0196] (e) comparing the difference in the labelled ligand bound in step(b) and (d), such that the compound which causes the reduction inbinding in step (d) is considered to be an agonist or antagonist.

[0197] The polypeptides may be found to modulate a variety ofphysiological and pathological processes in a dose-dependent manner inthe above-described assays. Thus, the “functional equivalents” of thepolypeptides of the invention include polypeptides that exhibit any ofthe same modulatory activities in the above-described assays in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the polypeptides of the invention,preferably the “functional equivalents” will exhibit substantiallysimilar dose-dependence in a given activity assay compared to thepolypeptides of the invention.

[0198] In certain of the embodiments described above, simple bindingassays may be used, in which the adherence of a test compound to asurface bearing the polypeptide is detected by means of a label directlyor indirectly associated with the test compound or in an assay involvingcompetition with a labelled competitor. In another embodiment,competitive drug screening assays may be used, in which neutralisingantibodies that are capable of binding the polypeptide specificallycompete with a test compound for binding. In this manner, the antibodiescan be used to detect the presence of any test compound that possessesspecific binding affinity for the polypeptide.

[0199] Persons skilled in the art will be able to devise assays foridentifying modulators of a polypeptide of the invention. Of interest inthis regard is Lokker N A et al J Biol Chem 1997 December.26;272(52):33037-44 which reports an example of an assay to identifyantagonists (in this case neutralizing antibodies).

[0200] Assays may also be designed to detect the effect of added testcompounds on the production of mRNA encoding the polypeptide in cells.For example, an ELISA may be constructed that measures secreted orcell-associated levels of polypeptide using monoclonal or polyclonalantibodies by standard methods known in the art, and this can be used tosearch for compounds that may inhibit or enhance the production of thepolypeptide from suitably manipulated cells or tissues. The formation ofbinding complexes between the polypeptide and the compound being testedmay then be measured.

[0201] Assay methods that are also included within the terms of thepresent invention are those that involve the use of the genes andpolypeptides of the invention in overexpression or ablation assays. Suchassays involve the manipulation of levels of these genes/polypeptides incells and assessment of the impact of this manipulation event on thephysiology of the manipulated cells. For example, such experimentsreveal details of signalling and metabolic pathways in which theparticular genes/polypeptides are implicated, generate informationregarding the identities of polypeptides with which the studiedpolypeptides interact and provide clues as to methods by which relatedgenes and proteins are regulated.

[0202] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the polypeptide of interest (see International patentapplication WO84/03564). In this method, large numbers of differentsmall test compounds are synthesised on a solid substrate, which maythen be reacted with the polypeptide of the invention and washed. Oneway of immobilising the polypeptide is to use non-neutralisingantibodies. Bound polypeptide may then be detected using methods thatare well known in the art. Purified polypeptide can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques.

[0203] The polypeptide of the invention may be used to identifymembrane-bound or soluble receptors, through standard receptor bindingtechniques that are known in the art, such as ligand binding andcrosslinking assays in which the polypeptide is labelled with aradioactive isotope, is chemically modified, or is fused to a peptidesequence that facilitates its detection or purification, and incubatedwith a source of the putative receptor (for example, a composition ofcells, cell membranes, cell supernatants, tissue extracts, or bodilyfluids). The efficacy of binding may be measured using biophysicaltechniques such as surface plasmon resonance and spectroscopy. Bindingassays may be used for the purification and cloning of the receptor, butmay also identify agonists and antagonists of the polypeptide, thatcompete with the binding of the polypeptide to its receptor. Standardmethods for conducting screening assays are well understood in the art.

[0204] The invention also includes a screening kit useful in the methodsfor identifying agonists, antagonists, ligands, receptors, substrates,enzymes, that are described above.

[0205] The invention includes the agonists, antagonists, ligands,receptors, substrates and enzymes, and other compounds which modulatethe activity or antigenicity of the polypeptide of the inventiondiscovered by the methods that are described above.

[0206] The invention also provides pharmaceutical compositionscomprising a polypeptide, nucleic acid, ligand or compound of theinvention in combination with a suitable pharmaceutical carrier. Thesecompositions may be suitable as therapeutic or diagnostic reagents, asvaccines, or as other immunogenic compositions, as outlined in detailbelow.

[0207] According to the terminology used herein, a compositioncontaining a polypeptide, nucleic acid, ligand or compound [α]is“substantially free of” impurities [herein, Y] when at least 85% byweight of the total X+Y in the composition is X. Preferably, X comprisesat least about 90% by weight of the total of X+Y in the composition,more preferably at least about 95%, 98% or even 99% by weight.

[0208] The pharmaceutical compositions should preferably comprise atherapeutically effective amount of the polypeptide, nucleic acidmolecule, ligand, or compound of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate, or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any compound, the therapeutically effectivedose can be estimated initially either in cell culture assays, forexample, of neoplastic cells, or in animal models, usually mice,rabbits, dogs, or pigs. The animal model may also be used to determinethe appropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

[0209] The precise effective amount for a human subject will depend uponthe severity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05mg/kg to 10 mg/kg. Compositions may be administered individually to apatient or may be administered in combination with other agents, drugsor hormones.

[0210] A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier, for administration of a therapeutic agent. Suchcarriers include antibodies and other polypeptides, genes and othertherapeutic agents such as liposomes, provided that the carrier does notitself induce the production of antibodies harmful to the individualreceiving the composition, and which may be administered without unduetoxicity. Suitable carriers may be large, slowly metabolisedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

[0211] Pharmaceutically acceptable salts can be used therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulphates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable carriers is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0212] Pharmaceutically acceptable carriers in therapeutic compositionsmay additionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such compositions. Such carriers enable the pharmaceuticalcompositions to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, foringestion by the patient.

[0213] Once formulated, the compositions of the invention can beadministered directly to the subject. The subjects to be treated can beanimals; in particular, human subjects can be treated.

[0214] The pharmaceutical compositions utilised in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal or transcutaneousapplications (for example, see WO98/20734), subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, intravaginalor rectal means. Gene guns or hyposprays may also be used to administerthe pharmaceutical compositions of the invention. Typically, thetherapeutic compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared.

[0215] Direct delivery of the compositions will generally beaccomplished by injection, subcutaneously, intraperitoneally,intravenously or intramuscularly, or delivered to the interstitial spaceof a tissue. The compositions can also be administered into a lesion.Dosage treatment may be a single dose schedule or a multiple doseschedule.

[0216] If the activity of the polypeptide of the invention is in excessin a particular disease state, several approaches are available. Oneapproach comprises administering to a subject an inhibitor compound(antagonist) as described above, along with a pharmaceuticallyacceptable carrier in an amount effective to inhibit the function of thepolypeptide, such as by blocking the binding of ligands, substrates,enzymes, receptors, or by inhibiting a second signal, and therebyalleviating the abnormal condition. Preferably, such antagonists areantibodies. Most preferably, such antibodies are chimeric and/orhumanised to minimise their immunogenicity, as described previously.

[0217] In another approach, soluble forms of the polypeptide that retainbinding affinity for the ligand, substrate, enzyme, receptor, inquestion, may be administered. Typically, the polypeptide may beadministered in the form of fragments that retain the relevant portions.

[0218] In an alternative approach, expression of the gene encoding thepolypeptide can be inhibited using expression blocking techniques, suchas the use of antisense nucleic acid molecules (as described above),either internally generated or separately administered. Modifications ofgene expression can be obtained by designing complementary sequences orantisense molecules (DNA, RNA, or PNA) to the control, 5′ or regulatoryregions (signal sequence, promoters, enhancers and introns) of the geneencoding the polypeptide. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The complementary sequence orantisense molecule may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes. Sucholigonucleotides may be administered or may be generated in situ fromexpression in vivo.

[0219] In addition, expression of the polypeptide of the invention maybe prevented by using ribozymes specific to its encoding mRNA sequence.Ribozymes are catalytically active RNAs that can be natural or synthetic(see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4),527-33). Synthetic ribozymes can be designed to specifically cleavemRNAs at selected positions thereby preventing translation of the mRNAsinto functional polypeptide. Ribozymes may be synthesised with a naturalribose phosphate backbone and natural bases, as normally found in RNAmolecules. Alternatively the ribozymes may be synthesised withnon-natural backbones, for example, 2′-O-methyl RNA, to provideprotection from ribonuclease degradation and may contain modified bases.

[0220] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of non-traditional bases such asinosine, queosine and butosine, as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine anduridine which are not as easily recognised by endogenous endonucleases.

[0221] For treating abnormal conditions related to an under-expressionof the polypeptide of the invention and its activity, several approachesare also available. One approach comprises administering to a subject atherapeutically effective amount of a compound that activates thepolypeptide, i.e., an agonist as described above, to alleviate theabnormal condition. Alternatively, a therapeutic amount of thepolypeptide in combination with a suitable pharmaceutical carrier may beadministered to restore the relevant physiological balance ofpolypeptide.

[0222] Gene therapy may be employed to effect the endogenous productionof the polypeptide by the relevant cells in the subject. Gene therapy isused to treat permanently the inappropriate production of thepolypeptide by replacing a defective gene with a corrected therapeuticgene.

[0223] Gene therapy of the present invention can occur in vivo or exvivo. Ex vivo gene therapy requires the isolation and purification ofpatient cells, the introduction of a therapeutic gene and introductionof the genetically altered cells back into the patient. In contrast, invivo gene therapy does not require isolation and purification of apatient's cells.

[0224] The therapeutic gene is typically “packaged” for administrationto a patient. Gene delivery vehicles may be non-viral, such asliposomes, or replication-deficient viruses, such as adenovirus asdescribed by Berkner, K. L., in Curr. Top. Microbiol. Immunol., 158,39-66 (1992) or adeno-associated virus (AAV) vectors as described byMuzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) andU.S. Pat. No. 5,252,479. For example, a nucleic acid molecule encoding apolypeptide of the invention may be engineered for expression in areplication-defective retroviral vector. This expression construct maythen be isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding the polypeptide, suchthat the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo (see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics (1996), T Strachan and A P Read,BIOS Scientific Publishers Ltd).

[0225] Another approach is the administration of “naked DNA” in whichthe therapeutic gene is directly injected into the bloodstream or muscletissue.

[0226] In situations in which the polypeptides or nucleic acid moleculesof the invention are disease-causing agents, the invention provides thatthey can be used in vaccines to raise antibodies against the diseasecausing agent.

[0227] Vaccines according to the invention may either be prophylactic(ie. to prevent infection) or therapeutic (ie. to treat disease afterinfection). Such vaccines comprise immunising antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid, usually in combination withpharmaceutically-acceptable carriers as described above, which includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition. Additionally, thesecarriers may function as immunostimulating agents (“adjuvants”).Furthermore, the antigen or immunogen may be conjugated to a bacterialtoxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori,and other pathogens.

[0228] Since polypeptides may be broken down in the stomach, vaccinescomprising polypeptides are preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient, and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents.

[0229] The vaccine formulations of the invention may be presented inunit-dose or multi-dose containers. For example, sealed ampoules andvials and may be stored in a freeze-dried condition requiring only theaddition of the sterile liquid carrier immediately prior to use. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0230] Genetic delivery of antibodies that bind to polypeptidesaccording to the invention may also be effected, for example, asdescribed in International patent application WO98/55607.

[0231] The technology referred to as jet injection (see, for example,www.powderject.com) may also be useful in the formulation of vaccinecompositions.

[0232] A number of suitable methods for vaccination and vaccine deliverysystems are described in International patent application WO00/29428.

[0233] This invention also relates to the use of nucleic acid moleculesaccording to the present invention as diagnostic reagents. Detection ofa mutated form of the gene characterised by the nucleic acid moleculesof the invention which is associated with a dysfunction will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, orsusceptibility to a disease, which results from under-expression,over-expression or altered spatial or temporal expression of the gene.Individuals carrying mutations in the gene may be detected at the DNAlevel by a variety of techniques.

[0234] Nucleic acid molecules for diagnosis may be obtained from asubject's cells, such as from blood, urine, saliva, tissue biopsy orautopsy material. The genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR, ligase chain reaction(LCR), strand displacement amplification (SDA), or other amplificationtechniques (see Saiki et al., Nature, 324, 163-166 (1986); Bej, et al.,Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer etal., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology,8, 291-294 (1990)) prior to analysis.

[0235] In one embodiment, this aspect of the invention provides a methodof diagnosing a disease in a patient, comprising assessing the level ofexpression of a natural gene encoding a polypeptide according to theinvention and comparing said level of expression to a control level,wherein a level that is different to said control level is indicative ofdisease. The method may comprise the steps of:

[0236] a) contacting a sample of tissue from the patient with a nucleicacid probe under stringent conditions that allow the formation of ahybrid complex between a nucleic acid molecule of the invention and theprobe;

[0237] b) contacting a control sample with said probe under the sameconditions used in step a);

[0238] c) and detecting the presence of hybrid complexes in saidsamples;

[0239] wherein detection of levels of the hybrid complex in the patientsample that differ from levels of the hybrid complex in the controlsample is indicative of disease.

[0240] A further aspect of the invention comprises a diagnostic methodcomprising the steps of:

[0241] a) obtaining a tissue sample from a patient being tested fordisease;

[0242] b)isolating a nucleic acid molecule according to the inventionfrom said tissue sample; and

[0243] c) diagnosing the patient for disease by detecting the presenceof a mutation in the nucleic acid molecule which is associated withdisease.

[0244] To aid the detection of nucleic acid molecules in theabove-described methods, an amplification step, for example using PCR,may be included.

[0245] Deletions and insertions can be detected by a change in the sizeof the amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to labelled RNAof the invention or alternatively, labelled antisense DNA sequences ofthe invention. Perfectly-matched sequences can be distinguished frommismatched duplexes by RNase digestion or by assessing differences inmelting temperatures. The presence or absence of the mutation in thepatient may be detected by contacting DNA with a nucleic acid probe thathybridises to the DNA under stringent conditions to form a hybriddouble-stranded molecule, the hybrid double-stranded molecule having anunhybridised portion of the nucleic acid probe strand at any portioncorresponding to a mutation associated with disease; and detecting thepresence or absence of an unhybridised portion of the probe strand as anindication of the presence or absence of a disease-associated mutationin the corresponding portion of the DNA strand.

[0246] Such diagnostics are particularly useful for prenatal and evenneonatal testing.

[0247] Point mutations and other sequence differences between thereference gene and “mutant” genes can be identified by other well-knowntechniques, such as direct DNA sequencing or single-strandconformational polymorphism, (see Orita et al., Genomics, 5, 874-879(1989)).

[0248] For example, a sequencing primer may be used with double-strandedPCR product or a single-stranded template molecule generated by amodified PCR. The sequence determination is performed by conventionalprocedures with radiolabelled nucleotides or by automatic sequencingprocedures with fluorescent-tags. Cloned DNA segments may also be usedas probes to detect specific DNA segments. The sensitivity of thismethod is greatly enhanced when combined with PCR. Further, pointmutations and other sequence variations, such as polymorphisms, can bedetected as described above, for example, through the use ofallele-specific oligonucleotides for PCR amplification of sequences thatdiffer by single nucleotides.

[0249] DNA sequence differences may also be detected by alterations inthe electrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (for example, Myers etal., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc.Natl. Acad. Sci. USA (1985) 85: 4397-4401).

[0250] In addition to conventional gel electrophoresis and DNAsequencing, mutations such as microdeletions, aneuploidies,translocations, inversions, can also be detected by in situ analysis(see, for example, Keller et al., DNA Probes, 2nd Ed., Stockton Press,New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells canbe analysed for mutations without need for their isolation and/orimmobilisation onto a membrane. Fluorescence in situ hybridization(FISH) is presently the most commonly applied method and numerousreviews of FISH have appeared (see, for example, Trachuck et al.,Science, 250, 559-562 (1990), and Trask et al., Trends, Genet., 7,149-154 (1991)).

[0251] In another embodiment of the invention, an array ofoligonucleotide probes comprising a nucleic acid molecule according tothe invention can be constructed to conduct efficient screening ofgenetic variants, mutations and polymorphisms. Array technology methodsare well known and have general applicability and can be used to addressa variety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science (1996), Vol 274, pp 610-613).

[0252] In one embodiment, the array is prepared and used according tothe methods described in PCT application WO95/11995 (Chee et al);Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena,M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).Oligonucleotide pairs may range from two to over one million. Theoligomers are synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support. In another aspect, an oligonucleotide may be synthesizedon the surface of the substrate by using a chemical coupling procedureand an ink jet application apparatus, as described in PCT applicationWO95/25116 (Baldeschweiler et al). In another aspect, a “gridded” arrayanalogous to a dot (or slot) blot may be used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system, thermal, UV, mechanical or chemical bonding procedures.An array, such as those described above, may be produced by hand or byusing available devices (slot blot or dot blot apparatus), materials(any suitable solid support), and machines (including roboticinstruments), and may contain 8, 24, 96, 384, 1536 or 6144oligonucleotides, or any other number between two and over one millionwhich lends itself to the efficient use of commercially-availableinstrumentation.

[0253] In addition to the methods discussed above, diseases may bediagnosed by methods comprising determining, from a sample derived froma subject, an abnormally decreased or increased level of polypeptide ormRNA. Decreased or increased expression can be measured at the RNA levelusing any of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods.

[0254] Assay techniques that can be used to determine levels of apolypeptide of the present invention in a sample derived from a host arewell-known to those of skill in the art and are discussed in some detailabove (including radioimmunoassays, competitive-binding assays, WesternBlot analysis and ELISA assays). This aspect of the invention provides adiagnostic method which comprises the steps of: (a) contacting a ligandas described above with a biological sample under conditions suitablefor the formation of a ligand-polypeptide complex; and (b) detectingsaid complex.

[0255] Protocols such as ELISA, RIA, and FACS for measuring polypeptidelevels may additionally provide a basis for diagnosing altered orabnormal levels of polypeptide expression. Normal or standard values forpolypeptide expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably humans, withantibody to the polypeptide under conditions suitable for complexformation The amount of standard complex formation may be quantified byvarious methods, such as by photometric means.

[0256] Antibodies which specifically bind to a polypeptide of theinvention may be used for the diagnosis of conditions or diseasescharacterised by expression of the polypeptide, or in assays to monitorpatients being treated with the polypeptides, nucleic acid molecules,ligands and other compounds of the invention. Antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for the polypeptideinclude methods that utilise the antibody and a label to detect thepolypeptide in human body fluids or extracts of cells or tissues. Theantibodies may be used with or without modification, and may be labelledby joining them, either covalently or non-covalently, with a reportermolecule. A wide variety of reporter molecules known in the art may beused, several of which are described above.

[0257] Quantities of polypeptide expressed in subject, control anddisease samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease. Diagnostic assays may be used todistinguish between absence, presence, and excess expression ofpolypeptide and to monitor regulation of polypeptide levels duringtherapeutic intervention. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials or in monitoring the treatment of anindividual patient.

[0258] A diagnostic kit of the present invention may comprise:

[0259] (a) a nucleic acid molecule of the present invention;

[0260] (b) a polypeptide of the present invention; or

[0261] (c) a ligand of the present invention.

[0262] In one aspect of the invention, a diagnostic kit may comprise afirst container containing a nucleic acid probe that hybridises understringent conditions with a nucleic acid molecule according to theinvention; a second container containing primers useful for amplifyingthe nucleic acid molecule; and instructions for using the probe andprimers for facilitating the diagnosis of disease. The kit may furthercomprise a third container holding an agent for digesting unhybridisedRNA.

[0263] In an alternative aspect of the invention, a diagnostic kit maycomprise an array of nucleic acid molecules, at least one of which maybe a nucleic acid molecule according to the invention.

[0264] To detect polypeptide according to the invention, a diagnostickit may comprise one or more antibodies that bind to a polypeptideaccording to the invention; and a reagent useful for the detection of abinding reaction between the antibody and the polypeptide.

[0265] Such kits will be of use in diagnosing a disease orsusceptibility to disease, including, but not limited to, diseasesincluding, but not limited to, cell proliferative disorders,autoimmune/inflammatory disorders, cardiovascular disorders,neurological and psychiatric disorders, developmental disorders, geneticdisorders, metabolic disorders, infections and other pathologicalconditions. These diseases preferably include neoplasm, cancer, braintumour, glioma, bone tumor, lung tumor, breast tumour, prostate tumour,colon tumour, hemangioma, myeloproliferative disorder, leukemia,hematological disease, neutropenia, thrombocytopenia, angiogenesisdisorders, dermatological disease, ageing, wounds, burns, fibrosis,cardiovascular disease, restensosis, heart disease, peripheral vasculardisease, coronary artery disease, oedema, thromboembolism, dysmenorrhea,endometriosis, pre-eclampsia, lung disease, COPD, asthma bone disease,renal disease, glomerulonephritis, liver disease, Crohn's disease,gastritis, ulcerative colitis, ulcer, immune disorder, autoimmunedisease, arthritis, rheumatoid arthritis, psoriasis, epidermolysisbullosa, systemic lupus erythematosus, ankylosing spondylitis, Lymedisease, multiple sclerosis, neurodegeneration, stroke, brain/spinalcord injury, Alzheimer's disease, Parkinson's disease, motor neuronedisease, neuromuscular disease, HIV, AIDS, cytomegalovirus infection,fungal infection, ocular disorder, macular degeneration, glaucoma,diabetic retinopathy, ocular hypertension and other conditions in whichimmunoglobulin domain containing cell recognition molecules areimplicated.

[0266] Various aspects and embodiments of the present invention will nowbe described in more detail by way of example, with particular referenceto the INSP052 and INSP055 polypeptides.

[0267] It will be appreciated that modification of detail may be madewithout departing from the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0268]FIG. 1: Results from BLAST against NCBI non-redundant databaseusing full-length INSP052 polypeptide sequence.

[0269]FIG. 2: Alignment generated by BLAST between the full-lengthINSP052 polypeptide sequence and the closest related sequence, biliaryglycoprotein H (mouse).

[0270]FIG. 3: Results from BLAST against NCBI non-redundant databaseusing full-length INSP055 polypeptide sequence.

[0271]FIG. 4: Alignment generated by BLAST between the full-lengthINSP055 polypeptide sequence and the closest related sequence, biliaryglycoprotein H (mouse).

[0272]FIG. 5: Predicted nucleotide sequence of INSP052 with translationunderlined sequence denotes predicted signal peptide. Boxed sequencedenotes predicted transmembrane domain.

[0273]FIG. 6: INSP052 coding exon organization in genomic DNA.Bottom=INSP052.cDNA, 1251 bp. Top=chr11.genomic_DNA. Sequence encodingthe putative extracellular domain is underlined. Start and Stop codonsare in bold type.

[0274]FIG. 7: Nucleotide sequence and translation of cloned INSP052extracellular domain.

[0275] Underlined sequence denotes predicted signal peptide.

[0276]FIG. 8: Map of pENTR-INSP052-EC-6HIS

[0277]FIG. 9: Map of pEAK12d-INSP052-EC-6HIS

[0278]FIG. 10: % secreted TNF (see Example 4)

[0279]FIG. 11: % secreted IL-4 (see Example 4)

[0280]FIG. 12: % secreted IL-2 (see Example 4)

[0281]FIG. 13: FIGS. 13A and 13B show that INSP052EC-eletrotransferredanimals show a decrease in transaminase levels as compared to emptyvector control animals 8 hours after the ConA challenge. (see Example 5)

[0282]FIG. 14: TNF-alpha and IL-6 cytokine levels inINSP052EC-eletrotransferred animals (see Example 5)

[0283]FIG. 15: ASAT and ALAT levels after 8 hours (see Example 5)

EXAMPLES Example 1 INSP052 and INSP055

[0284] The polypeptide sequence derived from combining SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ IDNO:14 and SEQ ID NO:16 which represents the translation of consecutiveexons from INSP052 is derived from human genomic sequence. Thepolynucleotide and polypeptide sequences SEQ ID NO 17 and SEQ ID 18representing INSP055 are polynucleotide and polypeptide sequences of themouse orthologue of INSP052 respectively. The existence of a mouseorthologue supports the gene model for the human sequence INSP52.

[0285] INSP052 and INSP055 polypeptide sequences represented by SEQ IDNO 16 and SEQ ID NO 18, respectively, are predicted to contain signalpeptide sequences and a transmembrane spanning domain.

[0286] The polypeptide sequence derived from combining SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:11, SEQ ID NO:12 and SEQID NO:14 and SEQ ID NO:116 which represents the translation ofconsecutive exons from INSP052, was used as a BLAST query against theNCBI non-redundant Sequence database. The top ten matches are shown inFIG. 1, all of which are immunoglobulin domain containing proteins.

[0287]FIG. 2 shows the alignment of the INSP052 query sequence to thesequence of the highest matching known protein, biliary glycoprotein H(mouse).

[0288] The polypeptide sequence INSP055, was used as a BLAST queryagainst the NCBI non-redundant Sequence database. The top ten matchesare shown in FIG. 3. FIG. 4 shows the alignment of the INSP055 querysequence to the sequence of the highest matching known protein, biliaryglycoprotein H (mouse).

[0289] Expressed sequence tags (ESTs) representing the INSP052 andINSP055 transcripts in human and mouse originate from the following cDNAlibraries: brain, including cerebellum, cortex, hippocampus,hypothalamus, medulla oblongata; inner ear and breast. Transcripts arealso represented by ESTs from oligodendroglioma, glioblastoma andmultiple sclerosis lesions. This suggests that INSP052 can be clonedfrom the above tissues and may be associated with diseases of the abovetissues. Accordingly, the polypeptides, antibodies and other moietiesdescribed herein may have utility in the treating a disease in one ofthe above tissues.

Example 2 Cloning of the INSP052 Extracellular Domain by Exon Assembly

[0290] The INSP052 full length prediction encodes a type I membraneprotein of 416 amino acids, related to the VEGF/PDGF receptors,belonging to the immunoglobulin superfamily. The predicted nucleotidesequence, starting from the initiating ATG codon to the poly A tail is2025 nucleotides long (FIG. 5). The coding sequence (cds) spans 7 exons(FIG. 6). A putative signal sequence (encoding amino acids 1-33) islocated in exon 1. The sequence encoding the predicted transmembrane(TM) domain (amino acids 241 to 263) is located at the exon 3-4boundary.

[0291] The extracellular (EC) domain encoding amino acids 1-240 wascloned by exon assembly from genomic DNA. An overview of the exonassembly method is summarized below:

[0292] Individual exons 1, 2 and 3 were amplified from genomic DNA byPCR. The reverse primer for exon 3 also contained an 11 base overlapwith the 5′ sequence of exon 4.

[0293] Gel-purified exons were mixed and a 2nd PCR reaction wasperformed to amplify the re-assembled DNA.

[0294] The full length PCR product corresponding to the INSP052 ECdomain was gel-purified and subcloned sequentially into pDONR 201(Gateway entry vector) and pEAK12d (expression vector) using theInvitrogen Gateway™ methodology.

[0295] 1. Pcr Amplification of Exons Encoding the Extracellular Domainof INSP052 From Genomic DNA.

[0296] PCR primers were designed to amplify exons 1, 2 and 3individually (table 1). The forward primer for exon 1(INSP052-B1P-exon1F) also contains the partial sequence of the GatewayattB1 site (5′ GCAGGCTTC ) and a Kozak sequence (5′ GCCACC). The reverseprimer for exon 1 (INSP052-exon1R) has an overlap of 18 bases with exon2 at its 5′ end. The forward primer for exon 2 (INSP052-exon2F) has an18 bp overlap with exon 1 at its 5′ end. The reverse primer for exon 2(INSP052-exon2R ) has an overlap of 18 bases with exon 3 at its 5′ end.The forward primer for exon 3 (INSP052-exon3F) contains an 17 bp overlapwith exon 2 at its 5′ end. The reverse primer for exon 3(INSP052-exon3R) has an overlap of 11 bases with exon 4 at its 5′ end.

[0297] For amplification of INSP052 exon 1, the PCR reaction wasperformed in a final volume of 50 μl and contained 1.5 μl of humangenomic DNA (0.11 g/μl, Novagen cat. no. 69237). 2 μl of 5 mM dNTPs(Amersham Pharmacia Biotech), 6 μl of INSP052-B1P-exon1F (10 μM), 6 1 μlof INSP052-exon1R , 5 μl of 10×Pwo buffer and 0.5 μl of Pwo polymerase(5 U/μl) (Roche, cat. no. 1 644 955). The PCR conditions were 94° C. for2 min; 35 cycles of 94° C. for 30 s, 60° C. for 30 s and 72° C. for 1min; an additional elongation cycle of 72° C. for 5 min; and a holdingcycle of 4° C. Reaction products were loaded onto a 1.5% agarose gel(1×TAE) and PCR products of the correct size (118 bp) were gel-purifiedusing a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and elutedin 50 μl of elution buffer (Qiagen).

[0298] Exon 2 was amplified using the same reaction conditions withprimers INSP052-exon2F and INSP052-exon2R. PCR products of 378 bp weregel purified as above.

[0299] Exon 3 was amplified using the same reaction conditions withprimers INSP052-exon3F and INSP052-exon3R. PCR products of 321 bp weregel purified as above.

[0300] 2. Assembly of Extracellular Domain-Encoding Exons of INSP052

[0301] Exons 1, 2 and 3-4 were re-assembled in a PCR reaction containing5 μl of each gel purified exon, 2 μl of 5 mM dNTPs, 6 μl ofINSP052-B1P-exon1F (10 μM), 6 μl of INSP052-5HIS-R (10 μM), 5 μl of10×Pfu buffer, 14.5 μl H₂O and 0.5 μl Pfu polymerase (3 U/μl; Promegacat. no. M774B). The reaction conditions were: 94° C., 4 min; 10 cyclesof 94° C. for 30 s, 48° C. for 30 s and 70° C. for 2 min; 25 cycles of94° C. for 30 s, 52° C., for 30 s and 70° C. for 2 min ; an additionalelongation step of 70° C. for 10 min; and a holding cycle at 4° C.Reaction products were analysed on a 1.5% agarose gel (1×TAE). PCRproducts of the correct size (750 bp) were gel purified using a QiaquickGel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl ofelution buffer (Qiagen). The resultant product (INSP052 EC ORF) containsthe ORF of the INSP052 EC domain flanked at the 5′ end by an attB1 siteand Kozak sequence, and at the 3′ end by a 5HIS tag encoding sequence.

[0302] 3. Subcloning of the INSP052 EC Domain ORF into pDONR201

[0303] AttB1 and attB2 recombination sites were added to the 5′ and 3′end of the full length INSP052 EC domain sequence in a PCR reactioncontaining 2 μl of gel purified INSP052 EC ORF, 2 μl of 5 mM dNTPs(Amersham Pharmacia Biotech), 6 μl of GCP-Forward (10 μM), 6 μl ofGCP-Reverse (10 μM), 5 μl of 10×Vent buffer and 0.5 μl of Vent DNApolymerase (2 U/μl) (New England Biolabs, cat. no. M0254S) in a finalvolume of 50 μl. The PCR conditions were 94° C. for 2 min; 30 cycles of94° C. for 30 sec; 55° C. for 30 sec and 72° C. for 1 min; an additionalelongation step of 72° C. for 3 min and a holding cycle of 4° C.Reaction products were analysed on a 1.5% agarose gel (1×TAE) and PCRproducts of the correct size (808 bp) were gel purified using a QiaquickGel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl ofelution buffer. (Qiagen). The purified PCR product (Gateway-modifiedINSP052 EC domain) was then transferred to pDONR201 using BP clonase asfollows: 5 μl of Gateway-modified INSP052 EC domain was incubated with1.5 μl pDONR201 (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonaseenzyme mix (Invitrogen) at RT for 1 h. The reaction was stopped byaddition of proteinase K (2 μg) and incubated at 37° C. for a further 10min. An aliquot of this reaction (1 μl) was transformed into 20 μl of E.coli DH10B cells (diluted ⅕ in H₂O) by electroporation using a BioradGene Pulser. Electroporated cells were diluted by addition of 1 ml ofSOC medium and incubated for 1 h at 37° C. Transformants were plated onLB-kanamycin plates and incubated overnight at 37° C. Plasmid mini prepDNA was isolated from 1-10 resultant colonies using a Qiaprep Turbo 9600robotic system (Qiagen) and subjected to DNA sequencing with pENTR-Fland pENTR-R1 sequencing primers using the BigDyeTerminator system(Applied Biosystems cat. no. 4390246) according to the manufacturer'sinstructions. Sequencing reactions were purified using Dye-Ex columns(Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624)then analyzed on an Applied Biosystems 3700 sequencer.

[0304] 4. Subcloning of the INSP052 EC Domain ORF to Expression VectorpEAK12d

[0305] Plasmid eluate (1.5 μl) from a pDONR201 clone containing thecorrect sequence of the INSP052 EC domain (plasmid ID # 13497) was thenused in a recombination reaction containing 1.5 μl pEAK12d (0.1 μg/μl),2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volumeof 10 μl. The mixture was incubated at RT for 1 h, stopped by additionof proteinase K (2 μg) and incubated at 37° C. for a further 10 min. Analiquot of this reaction (1 μl) was used to transform E. coli DH10Bcells by electroporation as described above. Electroporated cells werediluted by addition of 1 ml of SOC medium and incubated for 1 h at 37°C. Transformants were plated on LB-ampicillin plates and incubatedovernight at 37° C. Mini prep DNA was prepared from 4 colonies using aQiaprep Turbo 9600 robotic system (Qiagen) and eluted in 50 μl ofelution buffer. Two μl of each miniprep was then subjected to PCR in atotal reaction volume of 50 μl containing 2 μl of 5 mM dNTPs, 6 μl of 10μM pEAK12-F, 6 μl of 10 μM pEAK12-R, 5 μl of 10×AmpliTaq™ buffer and 0.5μl AmpliTaq™ (Applied Biosystems cat. no. N808-0155). The cyclingconditions were as follows: 94° C. for 2 min; 30 cycles of 94° C. for 30sec, 55° C. for 30 sec, and 72° C. for 1 min; 1 cycle, 72° C. for 3 min.Samples were then maintained at 4° C. (holding cycle) before furtheranalysis.

[0306] Plasmid mini prep DNA was isolated from colonies which gave theexpected PCR product size (1074 bp) was then subjected to DNA sequencingwith pEAK12-F and pEAK12-R sequencing primers.

[0307] CsCl gradient purified maxi-prep DNA of plasmidpEAK12d-INSP052EC-6HIS (plasmid ID # 13495) was prepared from a 500 mlculture of a sequence verified clone (Sambrook J. et al., in MolecularCloning, a Laboratory Manual, 2^(nd) edition, 1989, Cold Spring HarborLaboratory Press), resuspended at a concentration of 1 μg/μl in sterilewater and stored at −20 C. TABLE 2 Primers for INSP052 EC domain cloningand sequencing Primer Sequence (5′-3′) GCP Forward G GGG ACA AGT TTG TACAAA AAA GCA GGC TTC GCC ACC GCP Reverse GGG GAC CAC TTT GTA CAA GAA AGCTGG GTT TCA ATG GTG ATG GTG ATG GTG INSP052-B1P- GCA GGC TTC GCC ACC ATGAAG AGA GAA AGG GGA exon1F GCC CTG TC INSP052-exon1R TCA CCC CCT CCA GGGGGT CTG TCT GGA TCA GAA GAA INSP052-exon2F TTC TTC TGA TCC AGA CAG ACCCCC TGG AGG GGG TGA INSP052-exon2R GTG GCC TCG AAA TGG GCACAT CTA CAG TAA GGT TGA INSP052-exon3F CAA CCT TAC TGT AGA TGT GCC CATTTC GAG GCC ACA INSP052-exon3R GGA GCT TCT TCT GTA TAC GGT GAT CTT GACAG INSP052-5HIS-R GTG ATG GTG ATG GTG GGA GCT TCT TCT GTA TAC GGpEAK12-F GCC AGC TTG GCA CTT GAT GT pEAK12-R GAT GGA GGT GGA CGT GTC AGpENTR-F1 TCG CGT TAA CGC TAG CAT GGA TCT C pENTR-R1 GTA ACA TCA GAG ATTTTG AGA CAC

Example 3 Expression in Mammalian Cells of the Cloned, His-TaggedINSP052-6His-V1 (Plasmid No. 13495)

[0308] Human Embryonic Kidney 293 cells expressing the Epstein-Barrvirus Nuclear Antigen (HEK293-EBNA, Invitrogen) were maintained insuspension in Ex-cell VPRO serum-free medium (seed stock, maintenancemedium, JRH). Sixteen to 20 hours prior to transfection (Day-1), cellswere seeded in 2×T225 flasks (50 ml per flask in DMEM/F12 (1:1)containing 2% FBS seeding medium (JRH) at a density of 2×10⁵ cells/ml).The next day (transfection d a y 0) the transfection took place by usingthe JetPEI™ reagent (2 μl/μg of plasmid DNA, PolyPlus-transfection). Foreach flask, 113 μg of cDNA (plasmid No. 13495) was co-transfected with2.3 μg of GFP (fluorescent reporter gene). The transfection mix was thenadded to the 2×T225 flasks and incubated at 37° C. (5% CO₂) for 6 days.In order to increase our chances to get more material, we repeated thisprocedure into two extra flasks such as to generate 200 ml total.Confirmation of positive transfection was done by qualitativefluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss).

[0309] On day 6 (harvest day), supernatants (200 ml) from the fourflasks were pooled and centrifuged (4° C., 400 g) and placed into a potbearing a unique identifier.

[0310] One aliquot (500 ul) was kept for QC of the 6His-tagged protein(internal bioprocessing QC).

[0311] Purification Process

[0312] The 200 ml culture medium sample containing the recombinantprotein with a C-terminal 6His tag was diluted to a final volume of 200ml with cold buffer A (50 mM NaH₂PO₄; 600 mM NaCl; 8.7% (w/v) glycerol,pH 7.5). The sample was filtered through a 0.22 um sterile filter(Millipore, 500 ml filter unit) and kept at 4° C. in a 250 ml sterilesquare media bottle (Nalgene).

[0313] The purification was performed at 4° C. on the VISION workstation(Applied Biosystems) connected to an automatic sample loader(Labomatic). The purification procedure was composed of two sequentialsteps, metal affinity chromatography on a Poros 20 MC (AppliedBiosystems) column charged with Ni ions (4.6×50 mm, 0.83 ml), followedby gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column(1,0×10 cm).

[0314] For the first chromatography step the metal affinity column wasregenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 MNaCl; pH 8.0), recharged with Ni ions through washing with 15 columnvolumes of a 100 mM NiSO₄ solution, washed with 10 column volumes ofbuffer A, followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 600mM NaCl; 8.7% (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finallyequilibrated with 15 column volumes of buffer A containing 15 mMimidazole. The sample was transferred, by the Labomatic sample loader,into a 200 ml sample loop and subsequently charged onto the Ni metalaffinity column at a flow rate of 10 ml/min. The column was washed with12 column volumes of buffer A, followed by 28 column volumes of buffer Acontaining 20 mM imidazole. During the 20 mM imidazole wash looselyattached contaminating proteins were elution of the column. Therecombinant His-tagged protein was finally eluted with 10 column volumesof buffer B at a flow rate of 2 ml/min, and the eluted protein wascollected in a 1.6 ml fraction.

[0315] For the second chromatography step, the Sephadex G-25gel-filtration column was regenerated with 2 ml of buffer D (1.137 MNaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; pH 7.2), and subsequentlyequilibrated with 4 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl;1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 20% (w/v) glycerol; pH 7.4). The peakfraction eluted from the Ni-column was automatically through theintegrated sample loader on the VISION loaded onto the Sephadex G-25column and the protein was eluted with buffer C at a flow rate of 2ml/min. The desalted sample was recovered in a 2.2 ml fraction. Thefraction was filtered through a 0.22 um sterile centrifugation filter(Millipore), frozen and stored at −80C. An aliquot of the sample wasanalyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) by coomassie staining andWestern blot with anti-His antibodies.

[0316] Coomassie staining. The NuPAGE gel was stained in a 0.1%coomassie blue R250 staining solution (30% methanol, 10% acetic acid) atroom temperature for 1 h and subsequently destained in 20% methanol,7.5% acetic acid until the background was clear and the protein bandsclearly visible.

[0317] Western blot. Following the electrophoresis the proteins wereelectrotransferred from the gel to a nitrocellulose membrane at 290 mAfor 1 hour at 4° C. The membrane was blocked with 5% milk powder inbuffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 0.1%Tween 20, pH 7.4) for 1 h at room temperature, and subsequentlyincubated with a mixture of 2 rabbit polyclonal anti-His antibodies(G-18 and H-15; 0.2 ug/ml each; Santa Cruz) in 2.5% milk powder inbuffer E overnight at 4° C. After further 1 hour incubation at roomtemperature, the membrane was washed with buffer E (3×10 min), and thenincubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO,HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2hours at room temperature. After washing with buffer E (3×10 minutes),the membrane was developed with the ECL kit (Amersham Pharmacia) for 1min. The membrane was subsequently exposed to a Hyperfilm (AmershamPharmacia), the film developed and the western blot image visuallyanalyzed.

[0318] Protein assay. The protein concentration was determined using theBCA protein assay kit (Pierce) with bovine serum albumin as standard.890 μg purified protein was recovered from the 200 ml culture medium.

Example 4 Cytokine Expression Modulation Assays

[0319] 4.1 Introduction: The following in vitro cell-based assaysmeasure the effects of INSP052EC (cloned extracellular domain ofINSP052, see Examples 2 and 3) on cytokine secretion induced by fourdifferent stimuli on different human peripheral blood mononuclear cells(hPBMC) cells, as measured by a cytokine bead array (CBA) assay forIL-2, IFN-γ, TNF-α, IL-5, IL-4 and IL-10. Four different stimuli,Lipopolysaccharide (LPS), phytohemmagglutinin (PHA), Concanavalin A (ConA) and toxic shock syndrome toxin-1 (TSST-1), were used at 3 differentconcentrations for each at 3 different time points -24, 48 and 72 hours.

[0320] The best conditions are 100 000 cells/well in 96-well plates and100 μl final in 2% glycerol. The optimal concentration of mitogens are0.1 ng/ml for LPS, 1 ng/ml for PHA, 5 ng/ml for ConA and 0.1 ng/ml forTSST-1. The optimal time for the assay is 48 h. The optimalconcentration of the inhibitor, dexamethasone is 10-6 M. The optimalconcentration of the stimulator, hIL-18 is 100 ng/ml.

[0321] The read-out choice is the CBA.

[0322] 4.4.1 Purification of Human PBMC From a Buffy Coat

[0323] The buffy coat was diluted 1 to 2 with DMEM. 25 ml of dilutedblood was thereafter slowly added onto a 15 ml layer of Ficoll in a 50ml Falcon tube, and tubes were centrifuged (2000 rpm, 20 min, at RTwithout brake). The interphase (ring) was then collected and the cellswere washed with 25 ml of DMEM followed by a centrifuge step (1200 rpm,5 min). This procedure was repeated three times. A buffy coat gaveapproximately 600×10⁶ total cells.

[0324] 4.4.2 Screening

[0325] 80 μl of 1.25×10⁶ cells/ml were diluted in DMEM+2.5% HumanSerum+1% L-Glutamine+1% Penicillin-Streptomycin and thereafter added toa 96 well microtiter plate.

[0326] 10 μl were added per well (one condition per well): Proteins werediluted in PBS+20% Glycerol (the final dilution of the proteins is{fraction (1/10)}).

[0327] 10 μl of the 4 stimuli were then added per well (one conditionper well):

[0328] ConA 50 μg/ml. (the final concentration of ConA is 5 μg/ml)

[0329] LPS 1 μg/ml (the final concentration of LPS is 0.1 μg/ml)

[0330] -PHA 10 μg/ml (the final concentration of PHA is 1 μg/ml)

[0331] TSST-11 μg/ml (the final concentration of TSST-1 is 0.1 μg/ml)

[0332] After 48 h, cell supernatants were collected and human cytokineswere measured by Human Th1/Th2 Cytokine CBA Kit Becton-Dickinson.

[0333] For further clarification the Table below shows the experimentaldesign. 1 2 3 4 5 6 7 8 9 10 11 12 A Medium Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + Medium STIM STIM STIM STIMSTIM STIM STIM STIM STIM STIM B Medium Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + ConA STIM STIM STIM STIM STIMSTIM STIM STIM STIM STIM 5 μg/ml C STIM Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + ConA STIM STIM STIM STIM STIMSTIM STIM STIM STIM STIM 5 μg/ml D STIM Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + ConA STIM STIM STIM STIM STIMSTIM STIM STIM STIM STIM 5 μg/ml E STIM Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + ConA STIM STIM STIM STIM STIMSTIM STIM STIM STIM STIM 5 μg/ml F STIM + Prot + Prot + Prot + Prot +Prot + Prot + Prot + Prot + Prot + Prot + STIM + dexa STIM STIM STIMSTIM STIM STIM STIM STIM STIM STIM IL-18 10-6 M 100 ng/ml G STIM +Prot + Prot + Prot + Prot + Prot + Prot + Prot + Prot + Prot + Prot +STIM + dexa STIM STIM STIM STIM STIM STIM STIM STIM STIM STIM IL-18 10-6M 100 ng/ml H STIM + Prot + Prot + Prot + Prot + Prot + Prot + Prot +Prot + Prot + Prot + STIM + dexa STIM STIM STIM STIM STIM STIM STIM STIMSTIM STIM IL-18 10-6 M 100 ng/ml

[0334] 4.4.3 CBA Analysis

[0335] i) Preparation of Mixed Human Th1/Th2 Capture Beads

[0336] The number of assay tubes that were required for the experimentwas determined.

[0337] Each capture bead suspension was vigorously vortexed for a fewseconds before mixing. For each assay to be analysed, 10 μl aliquot ofeach capture bead were added into a single tube labelled “mixed capturebeads”. The Bead mixture was thoroughly vortexed.

[0338] ii) Preparation of Test Samples

[0339] Supernatants were diluted (1:4) using the Assay Diluent (20 μl ofsupernatants +60 μl of Assay Diluent). The sample dilution was thenmixed before transferring samples into a 96 wells microtiter plateconical bottom (Nunc).

[0340] iii) Human Th1/Th2 Cytokine CBA Assay Procedure

[0341] 50 μl of the diluted supernatants were added into a 96 wellsmicrotiter plate conical bottom (Nunc). 50 μl of the mixed capture beadswere added followed by 50 μl addition of the Human Th1/Th2 PE DetectionReagent. The plate was then incubated for 3 hours at RT and protectedfrom direct exposure to light followed by centrifugation at 1500 rpm for5 minutes. The supernatant was then carefully discarded. In a subsequentstep, 200 μl of wash buffer were twice added to each well, centrifugedat 1500 rpm for 5 minutes and supernatant carefully discarded. 130 μl ofwash buffer were thereafter added to each well to resuspend the beadpellet. The samples were finally analysed on a flow cytometer. The datawere analysed using the CBA Application Software, Activity Base andMicrosoft Excel software.

[0342] 4.5 Results

[0343] As shown in FIGS. 10, 11 and 12, INSP052EC was able todown-regulate in a dose-dependent manner the cytokine (TNF-alpha, IL-4and IL-2) secretion from ConA-stimulated hPBMC. In the FIGS. 10 to 12two different lots of the protein were tested. These results confirm apotential therapeutic efficacy of INSP052EC in the treatment ofanti-inflammatory and auto-immune diseases.

Example 5 Mouse Model of Fulminant Liver Hepatitis

[0344] 5.1 Introduction

[0345] Since INSP052EC protein has been shown in vitro to inhibitsecretion of certain cytokines by ConA-stimulated human peripheral bloodmononuclear cells (hPBMC) (see Example 4), it has been decided to testthe activity of INSP052EC in the in vivo ConA model by electrotransfer.

[0346] 5.2 Background—Concanavalin A (ConA)-Induced Liver Hepatitis

[0347] Toxic liver disease represents a worldwide health problem inhumans for which pharmacological treatments have yet to be discovered.For instance active chronic hepatitis leading to liver cirrhosis is adisease state, in which liver parenchymal cells are progressivelydestroyed by activated T cells. ConA-induced liver toxicity is one ofthree experimental models of T-cell dependent apoptotic and necroticliver injury described in mice. Gal N (D-Galactosamine) sensitized micechallenged with either activating anti-CD3 monoclonal AB or withsuperantigen SEB develop severe apoptotic and secondary necrotic liverinjury (Kusters S, Gastroenterology. 1996 August;111(2):462-71).Injection of the T-cell mitogenic plant lectin ConA to non sensitizedmice results also in hepatic apoptosis that preceeds necrosis. ConAinduces the release of systemic TNF-alpha and IFN-gamma and variousother cytokines. Both TNF-alpha and IFN-gamma are critical mediators ofliver injury. Transaminase release 8 hours after the insult indicatessevere liver destruction.

[0348] Several cell types have been shown to be involved in liverdamage, CD4 T cells, macrophages and natural killer cells (Kaneko J ExpMed 2000, 191, 105-114). Anti-CD4 antibodies block activation of T cellsand consequently liver damage (Tiegs et al. 1992, J Clin Invest 90,196-203). Pre-treatment of mice with monoclonal antibodies against CD8failed to protect, whereas deletion of macrophages prevented theinduction of hepatitis.

[0349] The present study was undertaken to investigate the role ofINSP052EC, a TNF-alpha antagonist protein containing IgG-like domains,in ConA-induced liver hepatitis. Several cytokines have been showneither to be critical in inducing or in conferring protection fromConA-induced liver damage. TNF-alpha for example is one of the firstcytokines produced after ConA injection and anti-TNF-alpha antibodiesconfer protection against disease (Seino et al. 2001, Annals of surgery234, 681). IFN-gamma appears also to be a critical mediator of liverinjury, since anti-IFN-gamma antiserum significantly protect mice, asmeasured by decreased levels of transaminases in the blood ofConA-treated animals (see Kusters et al., above). In liver injury,increased production of IFN-gamma was observed in patients withautoimmune or viral hepatitis. In addition transgenic mice expressingIFN-gamma in the liver develop liver injury resembling chronic activehepatitis (Toyonaga et al. 1994, PNAS 91, 614-618). IFN-gamma may alsobe cytotoxic to hepatocytes, since in vitro IFN-gamma induces cell deathin mouse hepatocytes that was accelerated by TNF (Morita et al. 1995,Hepatology 21, 1585-1593).

[0350] Other molecules have been described to be protective in the ConAmodel. A single administration of rhIL-6 completely inhibited therelease of transaminases (Mizuhara et al. 1994, J. Exp. Med. 179,1529-1537).

[0351] 5.3 cDNA Electrotransfer Into Muscle Fibers in Order to AchieveSystemic Expression of a Protein of Interest

[0352] Among the non-viral techniques for gene transfer in vivo, thedirect injection of plasmid DNA into the muscle and subsequentelectroporation is simple, inexpensive and safe. The post-mitotic natureand longevity of myofibers permits stable expression of transfectedgenes, although the transfected DNA does not usually undergo chromosomalintegration (Somiari et al. 2000, Molecular Therapy 2,178). Severalreports have demonstrated that secretion of muscle-produced proteinsinto the blood stream can be achieved after electroporation ofcorresponding cDNAs (Rizzuto et al. PNAS, 1996, 6417; Aihara H et al.,1998, Nature Biotech 16, 867). In addition in vivo efficacy of muscleexpressed Epo and IL-18BP in disease models has been shown (Rizzuto,2000, Human Gene Therapy 41, 1891; Mallat, 2001, !Circulation research89, 41).

[0353] 5.4 Materials and Methods

[0354] 5.4.1 Animals

[0355] In all the studies male C57/BL6 male (8 weeks of age) were used.In general, 7 animals per experimental group are used. Mice weremaintained in standard conditions under a 12-hour light-dark cycle,provided irradiated food and water ad libitum.

[0356] 5.4.2 Muscle Electrotransfer

[0357] 5.4.2.1 Choice of Vector

[0358] His or StrepII tagged IL6 and INSP052 genes were cloned in theGateway compatible pDEST12.2 vector containing the CMV promoter.

[0359] 5.4.2.2 Electroporation Protocol

[0360] Mice were anesthetized with gas (isofluran Baxter, Ref: ZDG9623).Hindlimbs were shaved and an echo graphic gel was applied. Hyaluronidasewas injected in the posterior tibialis mucle with (20 U in 50 μl sterileNaCl 0.9% , Sigma Ref. H3631). After 10 min, 100 μg of plasmid (50 μgper leg in 25 μl of sterile NaCl 0.9%) was injected in the same muscle.The DNA was prepared in the Buffer PBS-L-Glutamate (6 mg/ml; L-GlutamateSigma P4761) before intramuscular injection. For electrotransfer, theelectric field was applied for each leg with the ElectroSquarePoratorBTX ref ECM830 at 75 Volts during 20 ms for each pulse, 10 pulses withan interval of 1 second in a unipolar way with 2 round electrodes (size0.5 mm diameter).

[0361] 5.4.3 The ConA Model

[0362] 5.4.3.1 ConA i.v. Injection and Blood Sampling

[0363] 8 weeks old Female Mice C57/B16 were purchased from IFFA CREDO .ConA (Sigma ref.C7275) was injected at 18 mg/kg iv. and blood sampleswere taken at 1.30 and 8 hours postinjection. At the time of sacrifice,blood was taken from the heart.

[0364] 5.4.3.2 Detection of Cytokines and Transaminases in Blood Samples

[0365] IL2, IL5, IL4, TNF-alpha and IFN-gamma cytokine levels weremeasured using the TH1/TH2 CBA assay. TNF-alpha, IL-6, MCP1, IFN-alpha,IL-10 and IL-12 were detected using the Inflammation CBA assay.Transaminase blood parameters were determined using the COBAS instrument(Hitachi).

[0366] 5.4.3.3 INSP052EC and IL-6 Electrotransfer

[0367] At day 0 electrotransfer of pDEST12.2.—INSP052EC, pDEST12.2-hIL-6as well as and the empty vector control (electrotransfer protocol seeabove) was performed. At day 5 after electrotransfer, ConA (18 mg/kg)was injected i.v. and blood sampled at 2 time points (1.30, 8 hours).Cytokine and ASAT ALAT measurements were performed like describedabove).

[0368] 5.4.3.4 INSP052 and IL6 Protein Pretreatment in the ConA Model

[0369] CHO cell produced hIL-6 and HEK293 cell produced INSP052 wasinjected 30 min before ConA injection.

[0370] 5.5 Results

[0371] We have shown previously (see Example 4 and FIGS. 10-12) that HEK293 cell expressed INSP052EC protein down-regulates TNF-alpha and IL-4cytokine secretion in ConA and TSST-1 stimulated hPBMC in vitro in adose dependent way. Since these two cytokines play a crucial role in Tcell induced ConA induced liver hepatitis, we tested INSP052EC cDNA andprotein in this model.

[0372] We have now found that INSP052EC protects from liver injury in amouse model mimicking filminant hepatitis after systemic delivery of theprotein using electrotransfer. FIGS. 13A and 13B show thatINSP052EC-eletrotransferred animals show a decrease in transaminaseslevels as compared to empty vector control animals 8 hours after theConA challenge. In addition both TNF-alpha and IL-6 cytokine levels aresignificantly reduced in these animals (FIGS. 14A and 14B). Please notethat the effect is similar to that obtained with the positive controlvector pDEST12.2hIL-6-SII (FIGS. 14A and 14B).

[0373] In addition s.c. injected INSP052EC protein (1 mg/kg, 0.3 mg/kg)decreased ASAT and ALAT levels 8 hours after ConA injection (FIGS. 15Cand 15D).

[0374] 5.6 Conclusion

[0375] Our experiments have already shown, that INSP052EC downregulatesTNF-alpha, IL-4 and IL-2 secretion in vitro in the ConA stimulated hPBMCassay. In addition we could show that delivery of INSP052EC cDNA in anin vivo model of fulminant hepatitis decreases TNF-alpha and m-IL-6levels in serum and had a significant effect on the reduction oftransaminases measured in serum, which was confirmed by s.c. INSP052ECprotein injections.

[0376] The decrease in ASAT ALAT levels might be due to both, decreasedTNF-alpha and IL-4 levels. TNF-alpha and IL-4 are important cytokinesinvolved in the liver damage after ConA injection. In this mouse modelof liver hepatitis TNF-alpha is mainly produced by hepatic macrophages,the so-called Kupfer cells, whereas IL-4 is produced by liver (naturalkiller T) NKT cells. Anti TNF-alpha antibodies confer protection againstdisease (Seino et al. 2001, Annals of surgery 234, 681) and inhibitionof IL-4 production by NKT cells was shown to be hepato-protective inT-cell mediated hepatitis in mouse (Ajuebor et al. 2003 J. Immunology170, 5252-9).

[0377] INSP052EC might be useful in treating auto-immune, viral or acuteliver diseases as well as alcoholic liver failures. It might be alsoeffective in other inflammatory diseases.

[0378] The invention will now be described by the following numberedparagraphs:

[0379] 1. A polypeptide, which polypeptide:

[0380] (i) comprises or consists of the amino acid sequence as recitedin SEQ ID NO: 16 or SEQ ID NO:26;

[0381] (ii) is a fragment thereof having the activity of a polypeptideaccording to (i), or having an antigenic determinant in common with apolypeptide according to (i); or

[0382] (iii) is a functional equivalent of (i) or (ii).

[0383] 2. A polypeptide according to paragraph 1 part ii) whichcomprises or consists of the amino acid sequence as recited in SEQ IDNO:20 or in SEQ ID NO:22.

[0384] 3. A polypeptide which is a functional equivalent according toparagraph 1 (iii), characterised in that it is homologous to the aminoacid sequence as recited in SEQ ID NO: 16 or SEQ ID NO:26 and hasactivity as an antagonist of cytokine expression and/or secretion.

[0385] 4. A purified nucleic acid molecule which encodes a polypeptideaccording to any one of the preceding paragraphs.

[0386] 5. A purified nucleic acid molecule according to paragraph 4,which comprises the nucleic acid sequence as recited in SEQ ID NO:15,SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:25, or is a redundant equivalentor fragment thereof

[0387] 6. A purified nucleic acid molecule according to paragraph 5which consists of the nucleic acid sequence as recited in SEQ ID NO:15,SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:25.

[0388] 7. A purified nucleic acid molecule which hybridizes under highstringency conditions with a nucleic acid molecule according to any oneof paragraphs 4 to 6.

[0389] 8. A vector comprising a nucleic acid molecule as recited in anyone of paragraphs 4 to 7.

[0390] 9. A host cell transformed with a vector according to paragraph8.

[0391] 10. A ligand which binds specifically to, and which preferablyinhibits the activity of a polypeptide according to any one ofparagraphs 1 to 3.

[0392] 11. A ligand according to paragraph 10, which is an antibody.

[0393] 12. A compound that either increases or decreases the level ofexpression or activity of a polypeptide according to any one ofparagraphs 1 to 3.

[0394] 13. A compound according to paragraph 12 that binds to apolypeptide according to any one of paragraphs 1 to 3 without inducingany of the biological effects of the polypeptide.

[0395] 14. A compound according to paragraph 13, which is a natural ormodified substrate, ligand, enzyme, receptor or structural or functionalmimetic.

[0396] 15. A polypeptide according to any one of paragraphs 1 to 3, anucleic acid molecule according to any one of paragraphs 4 to 7, avector according to paragraph 8, a host cell according to paragraph 9, aligand according to paragraph 10 or paragraph 11, or a compoundaccording to any one of paragraphs 12 to 14, for use in therapy ordiagnosis of disease.

[0397] 16. A method of diagnosing a disease in a patient, comprisingassessing the level of expression of a natural gene encoding apolypeptide according to any one of paragraphs 1 to 3, or assessing theactivity of a polypeptide according to any one of paragraphs 1 to 3, intissue from said patient and comparing said level of expression oractivity to a control level, wherein a level that is different to saidcontrol level is indicative of disease.

[0398] 17. A method according to paragraph 16 that is carried out invitro.

[0399] 18. A method according to paragraph 16 or paragraph 17, whichcomprises the steps of: (a) contacting a ligand according to paragraph10 or paragraph 11 with a biological sample under conditions suitablefor the formation of a ligand-polypeptide complex; and (b) detectingsaid complex.

[0400] 19. A method according to paragraph 16 or paragraph 17,comprising the steps of:

[0401] a) contacting a sample of tissue from the patient with a nucleicacid probe under stringent conditions that allow the formation of ahybrid complex between a nucleic acid molecule according to any one ofparagraphs 4 to 7 and the probe;

[0402] b) contacting a control sample with said probe under the sameconditions used in step a); and

[0403] c) detecting the presence of hybrid complexes in said samples;wherein detection of levels of the hybrid complex in the patient samplethat differ from levels of the hybrid complex in the control sample isindicative of disease.

[0404] 20. A method according to paragraph 17 or paragraph 18,comprising:

[0405] a. contacting a sample of nucleic acid from tissue of the patientwith a nucleic acid primer under stringent conditions that allow theformation of a hybrid complex between a nucleic acid molecule accordingto any one of paragraphs 4 to 7 and the primer;

[0406] b. contacting a control sample with said primer under the sameconditions used in step a); and

[0407] c. amplifying the sampled nucleic acid; and

[0408] d. detecting the level of amplified nucleic acid from bothpatient and control samples; wherein detection of levels of theamplified nucleic acid in the patient sample that differ significantlyfrom levels of the amplified nucleic acid in the control sample isindicative of disease.

[0409] 21. A method according to paragraph 17 or paragraph 18comprising:

[0410] a. obtaining a tissue sample from a patient being tested fordisease;

[0411] b. isolating a nucleic acid molecule according to any one ofparagraphs 4 to 7 from said tissue sample; and

[0412] c. diagnosing the patient for disease by detecting the presenceof a mutation which is associated with disease in the nucleic acidmolecule as an indication of the disease.

[0413] 22. The method of paragraph 21, further comprising amplifying thenucleic acid molecule to form an amplified product and detecting thepresence or absence of a mutation in the amplified product.

[0414] 23. The method of paragraph 21 or paragraph 22, wherein thepresence or absence of the mutation in the patient is detected bycontacting said nucleic acid molecule with a nucleic acid probe thathybridises to said nucleic acid molecule under stringent conditions toform a hybrid double-stranded molecule, the hybrid double-strandedmolecule having an unhybridised portion of the nucleic acid probe strandat any portion corresponding to a mutation associated with disease; anddetecting the presence or absence of an unhybridised portion of theprobe strand as an indication of the presence or absence of adisease-associated mutation.

[0415] 24. A method according to any one of paragraphs 16 to 23, whereinsaid disease is an auto-immune, viral or acute liver disease, includingalcoholic liver failure, or inflammatory disease.

[0416] 25. Use of a polypeptide according to any one of paragraphs 1 to3 as an antagonist of cytokine expression and/or secretion.

[0417] 26. A pharmaceutical composition comprising a polypeptideaccording to any one of paragraphs 1 to 3, a nucleic acid moleculeaccording to any one of paragraphs 4 to 7, a vector according toparagraph 8, a host cell according to paragraph 9, a ligand according toparagraph 10 or paragraph 11, or a compound according to any one ofparagraphs 12 to 14.

[0418] 27. A vaccine composition comprising a polypeptide according toany one of paragraphs 1 to 3 or a nucleic acid molecule according to anyone of paragraphs 4 to 7.

[0419] 28. Use of a polypeptide according to any one of paragraphs 1 to3, a nucleic acid molecule according to any one of paragraphs 10 to 11,a vector according to paragraph 8, a host cell according to paragraph 9,a ligand according to paragraph 10 or paragraph 11, or a compoundaccording to any one of paragraphs 12 to 14 or a pharmaceuticalcomposition of paragraph 26, in the manufacture of a medicament for thetreatment of an auto-immune disease, viral or acute liver disease,including alcoholic liver failure, or inflammatory disease.

[0420] 29. A method of treating a disease in a patient, comprisingadministering to the patient a polypeptide according to any one ofparagraphs 1 to 3, a nucleic acid molecule according to any one ofparagraphs 4 to 7, a vector according to paragraph 8, a host cellaccording to paragraph 9, a ligand according to paragraph 10 orparagraph 11, or a compound according to any one of paragraphs 12 to 14or a pharmaceutical composition of paragraph 30.

[0421] 30. A method according to paragraph 29, wherein, for diseases inwhich the expression of the natural gene or the activity of thepolypeptide is lower in a diseased patient when compared to the level ofexpression or activity in a healthy patient, the polypeptide, nucleicacid molecule, vector, ligand, compound or composition administered tothe patient is an agonist.

[0422] 31. A method according to paragraph 29, wherein, for diseases inwhich the expression of the natural gene or activity of the polypeptideis higher in a diseased patient when compared to the level of expressionor activity in a healthy patient, the polypeptide, nucleic acidmolecule, vector, ligand, compound or composition administered to thepatient is an antagonist.

[0423] 32. A method of monitoring the therapeutic treatment of diseasein a patient, comprising monitoring over a period of time the level ofexpression or activity of a polypeptide according to any one ofparagraphs 1 to 3, or the level of expression of a nucleic acid moleculeaccording to any one of paragraphs 4 to 7 in tissue from said patient,wherein altering said level of expression or activity over the period oftime towards a control level is indicative of regression of saiddisease.

[0424] 33. A method for the identification of a compound that iseffective in the treatment and/or diagnosis of disease, comprisingcontacting a polypeptide according to any one of paragraphs 1 to 3, or anucleic acid molecule according to any one of paragraphs 4 to 7 with oneor more compounds suspected of possessing binding affinity for saidpolypeptide or nucleic acid molecule, and selecting a compound thatbinds specifically to said nucleic acid molecule or polypeptide.

[0425] 34. A kit useful for diagnosing disease comprising a firstcontainer containing a nucleic acid probe that hybridises understringent conditions with a nucleic acid molecule according to any oneof paragraphs 4 to 7; a second container containing primers useful foramplifying said nucleic acid molecule; and instructions for using theprobe and primers for facilitating the diagnosis of disease.

[0426] 35. The kit of paragraph 34, further comprising a third containerholding an agent for digesting unhybridised RNA.

[0427] 36. A kit comprising an array of nucleic acid molecules, at leastone of which is a nucleic acid molecule according to any one ofparagraphs 4 to 7.

[0428] 37. A kit comprising one or more antibodies that bind to apolypeptide as recited in any one of paragraphs 1 to 7, and a reagentuseful for the detection of a binding reaction between said antibody andsaid polypeptide.

[0429] 38. A transgenic or knockout non-human animal that has beentransformed to express higher, lower or absent levels of a polypeptideaccording to any one of paragraphs 1 to 3.

[0430] 39. A method for screening for a compound effective to treatdisease, by contacting a non-human transgenic animal according toparagraph 38 with a candidate compound and determining the effect of thecompound on the disease of the animal.

[0431] Sequence Information

[0432] Note: for amino acids encoded by exon-exon junctions, the aminoacid will be assigned to the more 5′ exon. SEQ ID NO 1: (INSP052Nucleotide sequence exon1) 1 ATGAAGAGAG AAAGGGGAGC CCTGTCCAGA GCCTCCAGGGCCCTGCGCCT TGCTCCTTTT 61 GTCTACCTTC TTCTGATCCA GACAG SEQ ID NO 2:(INSP052 polypeptide sequence of Exon 1) 1 MKRERGALSR ASRALRLAPFVYLLLIQTD SEQ Id NO 3: (INSP052 Nucleotide sequence exon2) 1 ACCCCCTGGAGGGGGTGAAC ATCACCAGCC CCGTGCGCCT GATCCATGGC ACCGTGGGGA 61 AGTCGGCTCTGCTTTCTGTG CAGTACAGCA GTACCAGCAG CGACAGGCCT GTAGTGAAGT 121 GGCAGCTGAAGCGGGACAAG CCAGTGACCG TGGTGCAGTC CATTGGCACA GAGGTCATCG 181 GCACCCTGCGGCCTGACTAT CGAGACCGTA TCCGACTCTT TGAAAATGGC TCCCTGCTTC 241 TCAGCGACCTGCAGCTGGCC GATGAGGGCA CCTATGAGGT CGAGATCTCC ATCACCGACG 301 ACACCTTCACTGGGGAGAAG ACCATCAACC TTACTGTAGA TG SEQ ID NO 4: (INSP052 ProteinSequence of Exon 2) 1 PLEGVNITSP VRLIHGTVGK SALLSVQYSS TSSDRPVVKWQLKRDKPVTV VQSIGTEVIG 61 TLRPDYRDRI RLFENGSLLL SDLQLADEGT YEVEISITDDTFTGEKTINL TVDV SEQ ID NO 5: (INSP052 Nucleotide sequence Exon3) 1TGCCCATTTC GAGGCCACAG GTGTTGGTGG CTTCAACCAC TGTGCTGGAG CTCAGCGAGG 61CCTTCACCTT GAACTGCTCA CATGAGAATG GCACCAAGCC CAGCTACACC TGGCTGAAGG 121ATGGCAAGCC CCTCCTCAAT GACTCGAGAA TGCTCCTGTC CCCCGACCAA AAGGTGCTCA 181CCATCACCCG CGTGCTCATG GAGGATGACG ACCTGTACAG CTGCATGGTG GAGAACCCCA 241TCAGCCAGGG CCGCAGCCTG CCTGTCAAGA TCACCGTATA CA SEQ ID NO 7: (INSP052Polypeptide sequence of Exon 3) 1 PISRPQVLVA STTVLELSEA FTLNCSHENGTKPSYTWLKD GKPLLNDSRM LLSPDQKVLT 61 ITRVLMEDDD LYSCMVENPI SQGRSLPVKITVYR SEQ ID NO 7: (INSP052 Nucleotide Sequence Exon 4) 1 GAAGAAGCTCCCTTTACATC ATCTTGTCTA CAGGAGGCAT CTTCCTCCTT GTGACCTTGG 61 TGACAGTCTGTGCCTGCTGG AAACCCTCCA AAAG SEQ ID NO 8: (INSP052 Polypeptide sequence ofExon 4) 1 RSSLYIILST GGIFLLVTLV TVCACWKPSK R SEQ ID NO 9: (INSP052Nucleotide Sequence Exon 5) 1 GAAACAGAAG AAGCTAGAAA AGCAAAACTCCCTGGAATAC ATGGATCAGA ATGATGACCG 61 CCTGAAACCA GAAG SEQ ID NO 10:(INSP052 Polypeptide Sequence Exon 5) 1 KQKKLEKQNS LEYMDQNDDR LKPEA SEQID NO 11: (INSP052 Nucleotide Sequence Exon 6) 1 CAGACACCCT CCCTCGAAGTGGTGAGCAGG AACGGAAGAA CCCCATGGCA CTCTATATCC 61 TGAAGGACAA G SEQ ID NO12: (INSP052 Polypeptide Sequence Exon 6) 1 DTLPRSGEQE RKNPMALYIL KDKSEQ ID NO 13: (INSP052 Nucleotide Sequence Exon 7) 1 GACTCCCCGGAGACCGAGGA GAACCCGGCC CCGGAGCCTC GAAGCGCGAC GGAGCCCGGC 61 CCGCCCGGCTACTCCGTGTC TCCCGCCGTG CCCGGCCGCT CGCCGGGGCT GCCCATCCGC 121 TCTGCCCGCCGCTACCCGCG CTCCCCAGCG CGCTCCCCAG CCACCGGCCG GACACACTCG 181 TCGCCGCCCAGGGCCCCGAG CTCGCCCGGC CGCTCGCGCA GCGCCTCGCG CACACTGCGG 241 ACTGCGGGCGTGCACATAAT CCGCGAGCAA GACGAGGCCG GCCCGGTGGA GATCAGCGCC 301 TGA SEQ ID NO14: (INSP052 Polypeptide sequence for exon 7) 1 DSPETEENPA PEPRSATEPGPPGYSVSPAV PGRSPGLPIR SARRYPRSPA RSPATGRTHS 61 SPPRAPSSPG RSRSASRTLRTAGVHIIREQ DEAGPVEISA SEQ ID NO :15 (INSP052 Combined Nucleotidesequence exons 1, 2, 3, 4, 5, 6 and 7) 1 ATGAAGAGAG AAAGGGGAGCCCTGTCCAGA GCCTCCAGGG CCCTGCGCCT TGCTCCTTTT 61 GTCTACCTTC TTCTGATCCAGACAGACCCC CTGGAGGGGG TGAACATCAC CAGCCCCGTG 121 CGCCTGATCC ATGGCACCGTGGGGAAGTCG GCTCTGCTTT CTGTGCAGTA CAGCAGTACC 181 AGCAGCGACA GGCCTGTAGTGAAGTGGCAG CTGAAGCGGG ACAAGCCAGT GACCGTGGTG 241 CAGTCCATTG GCACAGAGGTCATCGGCACC CTGCGGCCTG ACTATCGAGA CCGTATCCGA 301 CTCTTTGAAA ATGGCTCCCTGCTTCTCAGC GACCTGCAGC TGGCCGATGA GGGCACCTAT 361 GAGGTCGAGA TCTCCATCACCGACGACACC TTCACTGGGG AGAAGACCAT CAACCTTACT 421 GTAGATGTGC CCATTTCGAGGCCACAGGTG TTGGTGGCTT CAACCACTGT GCTGGAGCTC 481 AGCGAGGCCT TCACCTTGAACTGCTCACAT GAGAATGGCA CCAAGCCCAG CTACACCTGG 541 CTGAAGGATG GCAAGCCCCTCCTCAATGAC TCGAGAATGC TCCTGTCCCC CGACCAAAAG 601 GTGCTCACCA TCACCCGCGTGCTCATGGAG GATGACGACC TGTACAGCTG CATGGTGGAG 661 AACCCCATCA GCCAGGGCCGCAGCCTGCCT GTCAAGATCA CCGTATACAG AAGAAGCTCC 721 CTTTACATCA TCTTGTCTACAGGAGGCATC TTCCTCCTTG TGACCTTGGT GACAGTCTGT 781 GCCTGCTGGA AACCCTCCAAAAGGAAACAG AAGAAGCTAG AAAAGCAAAA CTCCCTGGAA 841 TACATGGATC AGAATGATGACCGCCTGAAA CCAGAAGCAG ACACCCTCCC TCGAAGTGGT 901 GAGCAGGAAC GGAAGAACCCCATGGCACTC TATATCCTGA AGGACAAGGA CTCCCCGGAG 961 ACCGAGGAGA ACCCGGCCCCGGAGCCTCGA AGCGCGACGG AGCCCGGCCC GCCCGGCTAC 1021 TCCGTGTCTC CCGCCGTGCCCGGCCGCTCG CCGGGGCTGC CCATCCGCTC TGCCCGCCGC 1081 TACCCGCGCT CCCCAGCGCGCTCCCCAGCC ACCGGCCGGA CACACTCGTC GCCGCCCAGG 1141 GCCCCGAGCT CGCCCGGCCGCTCGCGCAGC GCCTCGCGCA CACTGCGGAC TGCGGGCGTG 1201 CACATAATCC GCGAGCAAGACGAGGCCGGC CCGGTGGAGA TCAGCGCCTG A SEQ ID NO:16 (INSP052 Combinedpolypeptide sequence for exons 1, 2, 3, 4, 5, 6 and 7.) 1 MKRERGALSRASRALRLAPF VYLLLIQTDP LEGVNITSPV RLIHGTVGKS ALLSVQYSST 61 SSDRPVVKWQLKRDKPVTVV QSIGTEVIGT LRPDYRDRIR LFENGSLLLS DLQLADEGTY 121 EVEISITDDTFTGEKTINLT VDVPISRPQV LVASTTVLEL SEAFTLNCSH ENGTKPSYTW 181 LKDGKPLLNDSRMLLSPDQK VLTITRVLME DDDLYSCMVE NPISQGRSLP VKITVYRRSS 241 LYIILSTGGIFLLVTLVTVC ACWKPSKRKQ KKLEKQNSLE YMDQNDDRLK PEADTLPRSG 301 EQERKNPMALYILKDKDSPE TEENPAPEPR SATEPGPPGY SVSPAVPGRS PGLPIRSARR 361 YPRSPARSPATGRTHSSPPR APSSPGRSRS ASRTLRTAGV HIIREQDEAG PVEISA SEQ ID NO:17 (INSP055Mouse virtual cDNA) 1 ATGAAGAGAG AAAGGGGAGC CCTGTCAAGA GCCTCCAGGGCTCTGCGCCT CTCTCCTTTT 61 GTCTACCTGC TTCTCATCCA GCCAGTCCCC CTGGAGGGGGTGAACATCAC CAGCCCAGTA 121 CGTCTGATCC ACGGCACAGT GGGGAAGTCG GCCCTGCTTTCCGTGCAGTA CAGTAGCACC 181 AGCAGCGACA AGCCCGTGGT GAAGTGGCAG CTGAAGCGTGACAAGCCAGT GACCGTGGTG 241 CAGTCTATAG GCACAGAGGT CATTGGCACT CTGCGGCCTGACTATCGAGA CCGTATCCGG 301 CTCTTTGAAA ATGGCTCCTT GCTTCTCAGC GACCTGCAGCTGGCGGATGA GGGAACCTAT 361 GAAGTGGAGA TTTCCATCAC TGACGACACC TTCACCGGGGAGAAGACCAT CAACCTCACC 421 GTGGATGTGC CCATTTCAAG GCCGCAGGTA TTAGTGGCTTCAACCACTGT GCTGGAGCTC 481 AGTGAGGCCT TCACCCTCAA CTGCTCCCAT GAGAATGGCACCAAGCCTAG CTACACGTGG 541 CTGAAGGATG GCAAACCCCT CCTCAATGAC TCCCGAATGCTCCTGTCCCC TGACCAAAAG 601 GTGCTCACCA TCACCCGAGT ACTCATGGAA GATGACGACCTGTACAGCTG TGTGGTGGAG 661 AACCCCATCA GCCAGGTCCG CAGCCTGCCT GTCAAGATCACTGTGTATAG AAGAAGCTCC 721 CTCTATATCA TCTTGTCTAC AGGAGGCATC TTCCTCCTTGTGACCCTGGT GACAGTTTGT 781 GCCTGCTGGA AACCCTCAAA AAAGTCTAGG AAGAAGAGGAAGTTGGAGAA GCAAAACTCC 841 TTGGAATACA TGGATCAGAA TGATGACCGC CTAAAATCAGAAGCAGATAC CCTACCCCGA 901 AGTGGAGAAC AGGAGCGGAA GAACCCAATG GCACTCTATATCCTGAAGGA TAAGGATTCC 961 TCAGAGCCAG ATGAAAACCC TGCTACAGAG CCACGGAGCACCACAGAACC CGGTCCCCCT 1021 GGCTACTCCG TGTCGCCGCC CGTGCCCGGC CGCTCTCCGGGGCTTCCCAT CCGCTCAGCC 1081 CGCCGCTACC CGCGCTCCCC AGCACGTTCC CCTGCCACTGGCCGGACGCA CACGTCGCCA 1141 CCGCGGGCCC CGAGCTCGCC AGGCCGCTCG CGCAGCTCTTCGCGCTCACT GCGGACTGCA 1201 GGCGTGCAGA GAATCCGGGA GCAGGACGAG TCAGGGCAGGTGGAGATCAG TGCCTGA SEQ ID NO:18 (INSP055 Mouse Predicted Protein) 1MKRERGALSR ASRALRLSPF VYLLLIQPVP LEGVNITSPV RLIHGTVGKS ALLSVQYSST 61SSDKPVVKWQ LKRDKPVTVV QSIGTEVIGT LRPDYRDRIR LFENGSLLLS DLQLADEGTY 121EVEISITDDT FTGEKTINLT VDVPISRPQV LVASTTVLEL SEAFTLNCSH ENGTKPSYTW 181LKDGKPLLND SRMLLSPDQK VLTITRVLME DDDLYSCVVE NPISQVRSLP VKITVYRRSS 241LYIILSTGGI FLLVTLVTVC ACWKPSKKSR KKRKLEKQNS LEYMDQNDDR LKSEADTLPR 301SGEQERKNPM ALYILKDKDS SEPDENPATE PRSTTEPGPP GYSVSPPVPG RSPGLPIRSA 361RRYPRSPARS PATGRTHTSP PRAPSSPGRS RSSSRSLRTA GVQRIREQDE SGQVEISA SEQ IDNO:19 (nucleic acid sequence coding for extracellular domain of INSP052)1 ATGAAGAGAG AAAGGGGAGC CCTGTCCAGA GCCTCCAGGG CCCTGCGCCT TGCTCCTTTT 61GTCTACCTTC TTCTGATCCA GACAGACCCC CTGGAGGGGG TGAACATCAC CAGCCCCGTG 121CGCCTGATCC ATGGCACCGT GGGGAAGTCG GCTCTGCTTT CTGTGCAGTA CAGCAGTACC 181AGCAGCGACA GGCCTGTAGT GAAGTGGCAG CTGAAGCGGG ACAAGCCAGT GACCGTGGTG 241CAGTCCATTG GCACAGAGGT CATCGGCACC CTGCGGCCTG ACTATCGAGA CCGTATCCGA 301CTCTTTGAAA ATGGCTCCCT GCTTCTCAGC GACCTGCAGC TGGCCGATGA GGGCACCTAT 361GAGGTCGAGA TCTCCATCAC CGACGACACC TTCACTGGGG AGAAGACCAT CAACCTTACT 421GTAGATGTGC CCATTTCGAG GCCACAGGTG TTGGTGGCTT CAACCACTGT GCTGGAGCTC 481AGCGAGGCCT TCACCTTGAA CTGCTCACAT GAGAATGGCA CCAAGCCCAG CTACACCTGG 541CTGAAGGATG GCAAGCCCCT CCTCAATGAC TCGAGAATGC TCCTGTCCCC CGACCAAAAG 601GTGCTCACCA TCACCCGCGT GCTCATGGAG GATGACGACC TGTACAGCTG CATGGTGGAG 661AACCCCATCA GCCAGGGCCG CAGCCTGCCT GTCAAGATCA CCGTATACAG AAGAAGCTCC SEQ IDNO:20 (extracellular domain of INSP052) 1 MKRERGALSR ASRALRLAPFVYLLLIQTDP LEGVNITSPV RLIHGTVGKS ALLSVQYSST 61 SSDRPVVKWQ LKRDKPVTVVQSIGTEVIGT LRPDYRDRIR LFENGSLLLS DLQLADEGTY 121 EVEISITDDT FTGEKTINLTVDVPISRPQV LVASTTVLEL SEAFTLNCSH ENGTKPSYTW 181 LKDGKPLLND SRMLLSPDQKVLTITRVLME DDDLYSCMVE NPISQGRSLP VKITVYRRSS SEQ ID NO:21 (nucleic acidsequence coding for the extracellular domain of mature INSP052) GTGAACATCAC CAGCCCCGTG CGCCTGATCC ATGGCACCGT GGGGAAGTCG GCTCTGCTTTCTGTGCAGTA CAGCAGTACC AGCAGCGACA GGCCTGTAGT GAAGTGGCAG CTGAAGCGGGACAAGCCAGT GACCGTGGTG CAGTCCATTG GCACAGAGGT CATCGGCACC CTGCGGCCTGACTATCGAGA CCGTATCCGA CTCTTTGAAA ATGGCTCCCT GCTTCTCAGC GACCTGCAGCTGGCCGATGA GGGCACCTAT GAGGTCGAGA TCTCCATCAC CGACGACACC TTCACTGGGGAGAAGACCAT CAACCTTACT GTAGATGTGC CCATTTCGAG GCCACAGGTG TTGGTGGCTTCAACCACTGT GCTGGAGCTC AGCGAGGCCT TCACCTTGAA CTGCTCACAT GAGAATGGCACCAAGCCCAG CTACACCTGG CTGAAGGATG GCAAGCCCCT CCTCAATGAC TCGAGAATGCTCCTGTCCCC CGACCAAAAG GTGCTCACCA TCACCCGCGT GCTCATGGAG GATGACGACCTGTACAGCTG CATGGTGGAG AACCCCATCA GCCAGGGCCG CAGCCTGCCT GTCAAGATCACCGTATACAG AAGAAGCTCC SEQ ID NO:22 (extracellular domain of matureINSP052) VNITSPV RLIHGTVGKS ALLSVQYSST SSDRPVVKWQ LKRDKPVTVV QSIGTEVIGTLRPDYRDRIR LFENGSLLLS DLQLADEGTY EVEISITDDT FTGEKTINLT VDVPISRPQVLVASTTVLEL SEAFTLNCSH ENGTKPSYTW LKDGKPLLND SRMLLSPDQK VLTITRVLMEDDDLYSCMVE NPISQGRSLP VKITVYRRSS SEQ Id NO 23: (Nucleotide sequenceencoding the mature INSP052 exon2) GTGAAC ATCACCAGCC CCGTGCGCCTGATCCATGGC ACCGTGGGGA AGTCGGCTCT GCTTTCTGTG CAGTACAGCA GTACCAGCAGCGACAGGCCT GTAGTGAAGT GGCAGCTGAA GCGGGACAAG CCAGTGACCG TGGTGCAGTCCATTGGCACA GAGGTCATCG GCACCCTGCG GCCTGACTAT CGAGACCGTA TCCGACTCTTTGAAAATGGC TCCCTGCTTC TCAGCGACCT GCAGCTGGCC GATGAGGGCA CCTATGAGGTCGAGATCTCC ATCACCGACG ACACCTTCAC TGGGGAGAAG ACCATCAACC TTACTGTAGA TG SEQID NO 24: (Protein Sequence of Mature INSP052 Exon 2) 1 VNITSPVRLIHGTVGK SALLSVQYSS TSSDRPVVKW QLKRDKPVTV VQSIGTEVIG 61 TLRPDYRDRIRLFENGSLLL SDLQLADEGT YEVEISITDD TFTGEKTINL TVDV SEQ ID NO :25(nucleotide sequence encoding the mature INSP052 polypeptide) GTGAACATCAC CAGCCCCGTG CGCCTGATCC ATGGCACCGT GGGGAAGTCG GCTCTGCTTTCTGTGCAGTA CAGCAGTACC AGCAGCGACA GGCCTGTAGT GAAGTGGCAG CTGAAGCGGGACAAGCCAGT GACCGTGGTG CAGTCCATTG GCACAGAGGT CATCGGCACC CTGCGGCCTGACTATCGAGA CCGTATCCGA CTCTTTGAAA ATGGCTCCCT GCTTCTCAGC GACCTGCAGCTGGCCGATGA GGGCACCTAT GAGGTCGAGA TCTCCATCAC CGACGACACC TTCACTGGGGAGAAGACCAT CAACCTTACT GTAGATGTGC CCATTTCGAG GCCACAGGTG TTGGTGGCTTCAACCACTGT GCTGGAGCTC AGCGAGGCCT TCACCTTGAA CTGCTCACAT GAGAATGGCACCAAGCCCAG CTACACCTGG CTGAAGGATG GCAAGCCCCT CCTCAATGAC TCGAGAATGCTCCTGTCCCC CGACCAAAAG GTGCTCACCA TCACCCGCGT GCTCATGGAG GATGACGACCTGTACAGCTG CATGGTGGAG AACCCCATCA GCCAGGGCCG CAGCCTGCCT GTCAAGATCACCGTATACAG AAGAAGCTCC CTTTACATCA TCTTGTCTAC AGGAGGCATC TTCCTCCTTGTGACCTTGGT GACAGTCTGT GCCTGCTGGA AACCCTCCAA AAGGAAACAG AAGAAGCTAGAAAAGCAAAA CTCCCTGGAA TACATGGATC AGAATGATGA CCGCCTGAAA CCAGAAGCAGACACCCTCCC TCGAAGTGGT GAGCAGGAAC GGAAGAACCC CATGGCACTC TATATCCTGAAGGACAAGGA CTCCCCGGAG ACCGAGGAGA ACCCGGCCCC GGAGCCTCGA AGCGCGACGGAGCCCGGCCC GCCCGGCTAC TCCGTGTCTC CCGCCGTGCC CGGCCGCTCG CCGGGGCTGCCCATCCGCTC TGCCCGCCGC TACCCGCGCT CCCCAGCGCG CTCCCCAGCC ACCGGCCGGACACACTCGTC GCCGCCCAGG GCCCCGAGCT CGCCCGGCCG CTCGCGCAGC GCCTCGCGCACACTGCGGAC TGCGGGCGTG CACATAATCC GCGAGCAAGA CGAGGCCGGC CCGCTGGAGATCAGCGCCTG A SEQ ID NO:26 (INSP052 mature polypeptide sequence) VNITSPVRLIHGTVGKS ALLSVQYSST SSDRPVVKWQ LKRDKPVTVV QSIGTEVIGT LRPDYRDRIRLFENGSLLLS DLQLADEGTY EVEISITDDT FTGEKTINLT VDVPISRPQV LVASTTVLELSEAFTLNCSH ENGTKPSYTW LKDGKPLLND SRMLLSPDQK VLTITRVLME DDDLYSCMVENPISQGRSLP VKITVYRRSS LYIILSTGGI FLLVTLVTVC ACWKPSKRKQ KKLEKQNSLEYMDQNDDRLK PEADTLPRSG EQERKNPMAL YILKDKDSPE TEENPAPEPR SATEPGPPGYSVSPAVPGRS PGLPIRSARR YPRSPARSPA TGRTHSSPPR APSSPGRSRS ASRTLRTAGVHIIREQDEAG PVEISA

[0433]

1 43 1 85 DNA Homo sapiens 1 atgaagagag aaaggggagc cctgtccaga gcctccagggccctgcgcct tgctcctttt 60 gtctaccttc ttctgatcca gacag 85 2 29 PRT Homosapiens 2 Met Lys Arg Glu Arg Gly Ala Leu Ser Arg Ala Ser Arg Ala LeuArg 1 5 10 15 Leu Ala Pro Phe Val Tyr Leu Leu Leu Ile Gln Thr Asp 20 253 342 DNA Homo sapiens 3 accccctgga gggggtgaac atcaccagcc ccgtgcgcctgatccatggc accgtgggga 60 agtcggctct gctttctgtg cagtacagca gtaccagcagcgacaggcct gtagtgaagt 120 ggcagctgaa gcgggacaag ccagtgaccg tggtgcagtccattggcaca gaggtcatcg 180 gcaccctgcg gcctgactat cgagaccgta tccgactctttgaaaatggc tccctgcttc 240 tcagcgacct gcagctggcc gatgagggca cctatgaggtcgagatctcc atcaccgacg 300 acaccttcac tggggagaag accatcaacc ttactgtaga tg342 4 114 PRT Homo sapiens 4 Pro Leu Glu Gly Val Asn Ile Thr Ser Pro ValArg Leu Ile His Gly 1 5 10 15 Thr Val Gly Lys Ser Ala Leu Leu Ser ValGln Tyr Ser Ser Thr Ser 20 25 30 Ser Asp Arg Pro Val Val Lys Trp Gln LeuLys Arg Asp Lys Pro Val 35 40 45 Thr Val Val Gln Ser Ile Gly Thr Glu ValIle Gly Thr Leu Arg Pro 50 55 60 Asp Tyr Arg Asp Arg Ile Arg Leu Phe GluAsn Gly Ser Leu Leu Leu 65 70 75 80 Ser Asp Leu Gln Leu Ala Asp Glu GlyThr Tyr Glu Val Glu Ile Ser 85 90 95 Ile Thr Asp Asp Thr Phe Thr Gly GluLys Thr Ile Asn Leu Thr Val 100 105 110 Asp Val 5 282 DNA Homo sapiens 5tgcccatttc gaggccacag gtgttggtgg cttcaaccac tgtgctggag ctcagcgagg 60ccttcacctt gaactgctca catgagaatg gcaccaagcc cagctacacc tggctgaagg 120atggcaagcc cctcctcaat gactcgagaa tgctcctgtc ccccgaccaa aaggtgctca 180ccatcacccg cgtgctcatg gaggatgacg acctgtacag ctgcatggtg gagaacccca 240tcagccaggg ccgcagcctg cctgtcaaga tcaccgtata ca 282 6 94 PRT Homo sapiens6 Pro Ile Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr Val Leu Glu 1 5 1015 Leu Ser Glu Ala Phe Thr Leu Asn Cys Ser His Glu Asn Gly Thr Lys 20 2530 Pro Ser Tyr Thr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn Asp Ser 35 4045 Arg Met Leu Leu Ser Pro Asp Gln Lys Val Leu Thr Ile Thr Arg Val 50 5560 Leu Met Glu Asp Asp Asp Leu Tyr Ser Cys Met Val Glu Asn Pro Ile 65 7075 80 Ser Gln Gly Arg Ser Leu Pro Val Lys Ile Thr Val Tyr Arg 85 90 7 94DNA Homo sapiens 7 gaagaagctc cctttacatc atcttgtcta caggaggcatcttcctcctt gtgaccttgg 60 tgacagtctg tgcctgctgg aaaccctcca aaag 94 8 31PRT Homo sapiens 8 Arg Ser Ser Leu Tyr Ile Ile Leu Ser Thr Gly Gly IlePhe Leu Leu 1 5 10 15 Val Thr Leu Val Thr Val Cys Ala Cys Trp Lys ProSer Lys Arg 20 25 30 9 74 DNA Homo sapiens 9 gaaacagaag aagctagaaaagcaaaactc cctggaatac atggatcaga atgatgaccg 60 cctgaaacca gaag 74 10 25PRT Homo sapiens 10 Lys Gln Lys Lys Leu Glu Lys Gln Asn Ser Leu Glu TyrMet Asp Gln 1 5 10 15 Asn Asp Asp Arg Leu Lys Pro Glu Ala 20 25 11 71DNA Homo sapiens 11 cagacaccct ccctcgaagt ggtgagcagg aacggaagaaccccatggca ctctatatcc 60 tgaaggacaa g 71 12 23 PRT Homo sapiens 12 AspThr Leu Pro Arg Ser Gly Glu Gln Glu Arg Lys Asn Pro Met Ala 1 5 10 15Leu Tyr Ile Leu Lys Asp Lys 20 13 303 DNA Homo sapiens 13 gactccccggagaccgagga gaacccggcc ccggagcctc gaagcgcgac ggagcccggc 60 ccgcccggctactccgtgtc tcccgccgtg cccggccgct cgccggggct gcccatccgc 120 tctgcccgccgctacccgcg ctccccagcg cgctccccag ccaccggccg gacacactcg 180 tcgccgcccagggccccgag ctcgcccggc cgctcgcgca gcgcctcgcg cacactgcgg 240 actgcgggcgtgcacataat ccgcgagcaa gacgaggccg gcccggtgga gatcagcgcc 300 tga 303 14100 PRT Homo sapiens 14 Asp Ser Pro Glu Thr Glu Glu Asn Pro Ala Pro GluPro Arg Ser Ala 1 5 10 15 Thr Glu Pro Gly Pro Pro Gly Tyr Ser Val SerPro Ala Val Pro Gly 20 25 30 Arg Ser Pro Gly Leu Pro Ile Arg Ser Ala ArgArg Tyr Pro Arg Ser 35 40 45 Pro Ala Arg Ser Pro Ala Thr Gly Arg Thr HisSer Ser Pro Pro Arg 50 55 60 Ala Pro Ser Ser Pro Gly Arg Ser Arg Ser AlaSer Arg Thr Leu Arg 65 70 75 80 Thr Ala Gly Val His Ile Ile Arg Glu GlnAsp Glu Ala Gly Pro Val 85 90 95 Glu Ile Ser Ala 100 15 1251 DNA Homosapiens 15 atgaagagag aaaggggagc cctgtccaga gcctccaggg ccctgcgccttgctcctttt 60 gtctaccttc ttctgatcca gacagacccc ctggaggggg tgaacatcaccagccccgtg 120 cgcctgatcc atggcaccgt ggggaagtcg gctctgcttt ctgtgcagtacagcagtacc 180 agcagcgaca ggcctgtagt gaagtggcag ctgaagcggg acaagccagtgaccgtggtg 240 cagtccattg gcacagaggt catcggcacc ctgcggcctg actatcgagaccgtatccga 300 ctctttgaaa atggctccct gcttctcagc gacctgcagc tggccgatgagggcacctat 360 gaggtcgaga tctccatcac cgacgacacc ttcactgggg agaagaccatcaaccttact 420 gtagatgtgc ccatttcgag gccacaggtg ttggtggctt caaccactgtgctggagctc 480 agcgaggcct tcaccttgaa ctgctcacat gagaatggca ccaagcccagctacacctgg 540 ctgaaggatg gcaagcccct cctcaatgac tcgagaatgc tcctgtcccccgaccaaaag 600 gtgctcacca tcacccgcgt gctcatggag gatgacgacc tgtacagctgcatggtggag 660 aaccccatca gccagggccg cagcctgcct gtcaagatca ccgtatacagaagaagctcc 720 ctttacatca tcttgtctac aggaggcatc ttcctccttg tgaccttggtgacagtctgt 780 gcctgctgga aaccctccaa aaggaaacag aagaagctag aaaagcaaaactccctggaa 840 tacatggatc agaatgatga ccgcctgaaa ccagaagcag acaccctccctcgaagtggt 900 gagcaggaac ggaagaaccc catggcactc tatatcctga aggacaaggactccccggag 960 accgaggaga acccggcccc ggagcctcga agcgcgacgg agcccggcccgcccggctac 1020 tccgtgtctc ccgccgtgcc cggccgctcg ccggggctgc ccatccgctctgcccgccgc 1080 tacccgcgct ccccagcgcg ctccccagcc accggccgga cacactcgtcgccgcccagg 1140 gccccgagct cgcccggccg ctcgcgcagc gcctcgcgca cactgcggactgcgggcgtg 1200 cacataatcc gcgagcaaga cgaggccggc ccggtggaga tcagcgcctg a1251 16 416 PRT Homo sapiens 16 Met Lys Arg Glu Arg Gly Ala Leu Ser ArgAla Ser Arg Ala Leu Arg 1 5 10 15 Leu Ala Pro Phe Val Tyr Leu Leu LeuIle Gln Thr Asp Pro Leu Glu 20 25 30 Gly Val Asn Ile Thr Ser Pro Val ArgLeu Ile His Gly Thr Val Gly 35 40 45 Lys Ser Ala Leu Leu Ser Val Gln TyrSer Ser Thr Ser Ser Asp Arg 50 55 60 Pro Val Val Lys Trp Gln Leu Lys ArgAsp Lys Pro Val Thr Val Val 65 70 75 80 Gln Ser Ile Gly Thr Glu Val IleGly Thr Leu Arg Pro Asp Tyr Arg 85 90 95 Asp Arg Ile Arg Leu Phe Glu AsnGly Ser Leu Leu Leu Ser Asp Leu 100 105 110 Gln Leu Ala Asp Glu Gly ThrTyr Glu Val Glu Ile Ser Ile Thr Asp 115 120 125 Asp Thr Phe Thr Gly GluLys Thr Ile Asn Leu Thr Val Asp Val Pro 130 135 140 Ile Ser Arg Pro GlnVal Leu Val Ala Ser Thr Thr Val Leu Glu Leu 145 150 155 160 Ser Glu AlaPhe Thr Leu Asn Cys Ser His Glu Asn Gly Thr Lys Pro 165 170 175 Ser TyrThr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn Asp Ser Arg 180 185 190 MetLeu Leu Ser Pro Asp Gln Lys Val Leu Thr Ile Thr Arg Val Leu 195 200 205Met Glu Asp Asp Asp Leu Tyr Ser Cys Met Val Glu Asn Pro Ile Ser 210 215220 Gln Gly Arg Ser Leu Pro Val Lys Ile Thr Val Tyr Arg Arg Ser Ser 225230 235 240 Leu Tyr Ile Ile Leu Ser Thr Gly Gly Ile Phe Leu Leu Val ThrLeu 245 250 255 Val Thr Val Cys Ala Cys Trp Lys Pro Ser Lys Arg Lys GlnLys Lys 260 265 270 Leu Glu Lys Gln Asn Ser Leu Glu Tyr Met Asp Gln AsnAsp Asp Arg 275 280 285 Leu Lys Pro Glu Ala Asp Thr Leu Pro Arg Ser GlyGlu Gln Glu Arg 290 295 300 Lys Asn Pro Met Ala Leu Tyr Ile Leu Lys AspLys Asp Ser Pro Glu 305 310 315 320 Thr Glu Glu Asn Pro Ala Pro Glu ProArg Ser Ala Thr Glu Pro Gly 325 330 335 Pro Pro Gly Tyr Ser Val Ser ProAla Val Pro Gly Arg Ser Pro Gly 340 345 350 Leu Pro Ile Arg Ser Ala ArgArg Tyr Pro Arg Ser Pro Ala Arg Ser 355 360 365 Pro Ala Thr Gly Arg ThrHis Ser Ser Pro Pro Arg Ala Pro Ser Ser 370 375 380 Pro Gly Arg Ser ArgSer Ala Ser Arg Thr Leu Arg Thr Ala Gly Val 385 390 395 400 His Ile IleArg Glu Gln Asp Glu Ala Gly Pro Val Glu Ile Ser Ala 405 410 415 17 1257DNA Homo sapiens 17 atgaagagag aaaggggagc cctgtcaaga gcctccagggctctgcgcct ctctcctttt 60 gtctacctgc ttctcatcca gccagtcccc ctggagggggtgaacatcac cagcccagta 120 cgtctgatcc acggcacagt ggggaagtcg gccctgctttccgtgcagta cagtagcacc 180 agcagcgaca agcccgtggt gaagtggcag ctgaagcgtgacaagccagt gaccgtggtg 240 cagtctatag gcacagaggt cattggcact ctgcggcctgactatcgaga ccgtatccgg 300 ctctttgaaa atggctcctt gcttctcagc gacctgcagctggcggatga gggaacctat 360 gaagtggaga tttccatcac tgacgacacc ttcaccggggagaagaccat caacctcacc 420 gtggatgtgc ccatttcaag gccgcaggta ttagtggcttcaaccactgt gctggagctc 480 agtgaggcct tcaccctcaa ctgctcccat gagaatggcaccaagcctag ctacacgtgg 540 ctgaaggatg gcaaacccct cctcaatgac tcccgaatgctcctgtcccc tgaccaaaag 600 gtgctcacca tcacccgagt actcatggaa gatgacgacctgtacagctg tgtggtggag 660 aaccccatca gccaggtccg cagcctgcct gtcaagatcactgtgtatag aagaagctcc 720 ctctatatca tcttgtctac aggaggcatc ttcctccttgtgaccctggt gacagtttgt 780 gcctgctgga aaccctcaaa aaagtctagg aagaagaggaagttggagaa gcaaaactcc 840 ttggaataca tggatcagaa tgatgaccgc ctaaaatcagaagcagatac cctaccccga 900 agtggagaac aggagcggaa gaacccaatg gcactctatatcctgaagga taaggattcc 960 tcagagccag atgaaaaccc tgctacagag ccacggagcaccacagaacc cggtccccct 1020 ggctactccg tgtcgccgcc cgtgcccggc cgctctccggggcttcccat ccgctcagcc 1080 cgccgctacc cgcgctcccc agcacgttcc cctgccactggccggacgca cacgtcgcca 1140 ccgcgggccc cgagctcgcc aggccgctcg cgcagctcttcgcgctcact gcggactgca 1200 ggcgtgcaga gaatccggga gcaggacgag tcagggcaggtggagatcag tgcctga 1257 18 418 PRT Homo sapiens 18 Met Lys Arg Glu ArgGly Ala Leu Ser Arg Ala Ser Arg Ala Leu Arg 1 5 10 15 Leu Ser Pro PheVal Tyr Leu Leu Leu Ile Gln Pro Val Pro Leu Glu 20 25 30 Gly Val Asn IleThr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly 35 40 45 Lys Ser Ala LeuLeu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Lys 50 55 60 Pro Val Val LysTrp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val 65 70 75 80 Gln Ser IleGly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg 85 90 95 Asp Arg IleArg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu 100 105 110 Gln LeuAla Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile Thr Asp 115 120 125 AspThr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val Asp Val Pro 130 135 140Ile Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr Val Leu Glu Leu 145 150155 160 Ser Glu Ala Phe Thr Leu Asn Cys Ser His Glu Asn Gly Thr Lys Pro165 170 175 Ser Tyr Thr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn Asp SerArg 180 185 190 Met Leu Leu Ser Pro Asp Gln Lys Val Leu Thr Ile Thr ArgVal Leu 195 200 205 Met Glu Asp Asp Asp Leu Tyr Ser Cys Val Val Glu AsnPro Ile Ser 210 215 220 Gln Val Arg Ser Leu Pro Val Lys Ile Thr Val TyrArg Arg Ser Ser 225 230 235 240 Leu Tyr Ile Ile Leu Ser Thr Gly Gly IlePhe Leu Leu Val Thr Leu 245 250 255 Val Thr Val Cys Ala Cys Trp Lys ProSer Lys Lys Ser Arg Lys Lys 260 265 270 Arg Lys Leu Glu Lys Gln Asn SerLeu Glu Tyr Met Asp Gln Asn Asp 275 280 285 Asp Arg Leu Lys Ser Glu AlaAsp Thr Leu Pro Arg Ser Gly Glu Gln 290 295 300 Glu Arg Lys Asn Pro MetAla Leu Tyr Ile Leu Lys Asp Lys Asp Ser 305 310 315 320 Ser Glu Pro AspGlu Asn Pro Ala Thr Glu Pro Arg Ser Thr Thr Glu 325 330 335 Pro Gly ProPro Gly Tyr Ser Val Ser Pro Pro Val Pro Gly Arg Ser 340 345 350 Pro GlyLeu Pro Ile Arg Ser Ala Arg Arg Tyr Pro Arg Ser Pro Ala 355 360 365 ArgSer Pro Ala Thr Gly Arg Thr His Thr Ser Pro Pro Arg Ala Pro 370 375 380Ser Ser Pro Gly Arg Ser Arg Ser Ser Ser Arg Ser Leu Arg Thr Ala 385 390395 400 Gly Val Gln Arg Ile Arg Glu Gln Asp Glu Ser Gly Gln Val Glu Ile405 410 415 Ser Ala 19 720 DNA Homo sapiens 19 atgaagagag aaaggggagccctgtccaga gcctccaggg ccctgcgcct tgctcctttt 60 gtctaccttc ttctgatccagacagacccc ctggaggggg tgaacatcac cagccccgtg 120 cgcctgatcc atggcaccgtggggaagtcg gctctgcttt ctgtgcagta cagcagtacc 180 agcagcgaca ggcctgtagtgaagtggcag ctgaagcggg acaagccagt gaccgtggtg 240 cagtccattg gcacagaggtcatcggcacc ctgcggcctg actatcgaga ccgtatccga 300 ctctttgaaa atggctccctgcttctcagc gacctgcagc tggccgatga gggcacctat 360 gaggtcgaga tctccatcaccgacgacacc ttcactgggg agaagaccat caaccttact 420 gtagatgtgc ccatttcgaggccacaggtg ttggtggctt caaccactgt gctggagctc 480 agcgaggcct tcaccttgaactgctcacat gagaatggca ccaagcccag ctacacctgg 540 ctgaaggatg gcaagcccctcctcaatgac tcgagaatgc tcctgtcccc cgaccaaaag 600 gtgctcacca tcacccgcgtgctcatggag gatgacgacc tgtacagctg catggtggag 660 aaccccatca gccagggccgcagcctgcct gtcaagatca ccgtatacag aagaagctcc 720 20 240 PRT Homo sapiens20 Met Lys Arg Glu Arg Gly Ala Leu Ser Arg Ala Ser Arg Ala Leu Arg 1 510 15 Leu Ala Pro Phe Val Tyr Leu Leu Leu Ile Gln Thr Asp Pro Leu Glu 2025 30 Gly Val Asn Ile Thr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly 3540 45 Lys Ser Ala Leu Leu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Arg 5055 60 Pro Val Val Lys Trp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val 6570 75 80 Gln Ser Ile Gly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg85 90 95 Asp Arg Ile Arg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu100 105 110 Gln Leu Ala Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile ThrAsp 115 120 125 Asp Thr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val AspVal Pro 130 135 140 Ile Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr ValLeu Glu Leu 145 150 155 160 Ser Glu Ala Phe Thr Leu Asn Cys Ser His GluAsn Gly Thr Lys Pro 165 170 175 Ser Tyr Thr Trp Leu Lys Asp Gly Lys ProLeu Leu Asn Asp Ser Arg 180 185 190 Met Leu Leu Ser Pro Asp Gln Lys ValLeu Thr Ile Thr Arg Val Leu 195 200 205 Met Glu Asp Asp Asp Leu Tyr SerCys Met Val Glu Asn Pro Ile Ser 210 215 220 Gln Gly Arg Ser Leu Pro ValLys Ile Thr Val Tyr Arg Arg Ser Ser 225 230 235 240 21 621 DNA Homosapiens 21 gtgaacatca ccagccccgt gcgcctgatc catggcaccg tggggaagtcggctctgctt 60 tctgtgcagt acagcagtac cagcagcgac aggcctgtag tgaagtggcagctgaagcgg 120 gacaagccag tgaccgtggt gcagtccatt ggcacagagg tcatcggcaccctgcggcct 180 gactatcgag accgtatccg actctttgaa aatggctccc tgcttctcagcgacctgcag 240 ctggccgatg agggcaccta tgaggtcgag atctccatca ccgacgacaccttcactggg 300 gagaagacca tcaaccttac tgtagatgtg cccatttcga ggccacaggtgttggtggct 360 tcaaccactg tgctggagct cagcgaggcc ttcaccttga actgctcacatgagaatggc 420 accaagccca gctacacctg gctgaaggat ggcaagcccc tcctcaatgactcgagaatg 480 ctcctgtccc ccgaccaaaa ggtgctcacc atcacccgcg tgctcatggaggatgacgac 540 ctgtacagct gcatggtgga gaaccccatc agccagggcc gcagcctgcctgtcaagatc 600 accgtataca gaagaagctc c 621 22 207 PRT Homo sapiens 22Val Asn Ile Thr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly Lys 1 5 1015 Ser Ala Leu Leu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Arg Pro 20 2530 Val Val Lys Trp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val Gln 35 4045 Ser Ile Gly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg Asp 50 5560 Arg Ile Arg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu Gln 65 7075 80 Leu Ala Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile Thr Asp Asp 8590 95 Thr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val Asp Val Pro Ile100 105 110 Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr Val Leu Glu LeuSer 115 120 125 Glu Ala Phe Thr Leu Asn Cys Ser His Glu Asn Gly Thr LysPro Ser 130 135 140 Tyr Thr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn AspSer Arg Met 145 150 155 160 Leu Leu Ser Pro Asp Gln Lys Val Leu Thr IleThr Arg Val Leu Met 165 170 175 Glu Asp Asp Asp Leu Tyr Ser Cys Met ValGlu Asn Pro Ile Ser Gln 180 185 190 Gly Arg Ser Leu Pro Val Lys Ile ThrVal Tyr Arg Arg Ser Ser 195 200 205 23 328 DNA Homo sapiens 23gtgaacatca ccagccccgt gcgcctgatc catggcaccg tggggaagtc ggctctgctt 60tctgtgcagt acagcagtac cagcagcgac aggcctgtag tgaagtggca gctgaagcgg 120gacaagccag tgaccgtggt gcagtccatt ggcacagagg tcatcggcac cctgcggcct 180gactatcgag accgtatccg actctttgaa aatggctccc tgcttctcag cgacctgcag 240ctggccgatg agggcaccta tgaggtcgag atctccatca ccgacgacac cttcactggg 300gagaagacca tcaaccttac tgtagatg 328 24 110 PRT Homo sapiens 24 Val AsnIle Thr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly Lys 1 5 10 15 SerAla Leu Leu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Arg Pro 20 25 30 ValVal Lys Trp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val Gln 35 40 45 SerIle Gly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg Asp 50 55 60 ArgIle Arg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu Gln 65 70 75 80Leu Ala Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile Thr Asp Asp 85 90 95Thr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val Asp Val 100 105 110 251152 DNA Homo sapiens 25 gtgaacatca ccagccccgt gcgcctgatc catggcaccgtggggaagtc ggctctgctt 60 tctgtgcagt acagcagtac cagcagcgac aggcctgtagtgaagtggca gctgaagcgg 120 gacaagccag tgaccgtggt gcagtccatt ggcacagaggtcatcggcac cctgcggcct 180 gactatcgag accgtatccg actctttgaa aatggctccctgcttctcag cgacctgcag 240 ctggccgatg agggcaccta tgaggtcgag atctccatcaccgacgacac cttcactggg 300 gagaagacca tcaaccttac tgtagatgtg cccatttcgaggccacaggt gttggtggct 360 tcaaccactg tgctggagct cagcgaggcc ttcaccttgaactgctcaca tgagaatggc 420 accaagccca gctacacctg gctgaaggat ggcaagcccctcctcaatga ctcgagaatg 480 ctcctgtccc ccgaccaaaa ggtgctcacc atcacccgcgtgctcatgga ggatgacgac 540 ctgtacagct gcatggtgga gaaccccatc agccagggccgcagcctgcc tgtcaagatc 600 accgtataca gaagaagctc cctttacatc atcttgtctacaggaggcat cttcctcctt 660 gtgaccttgg tgacagtctg tgcctgctgg aaaccctccaaaaggaaaca gaagaagcta 720 gaaaagcaaa actccctgga atacatggat cagaatgatgaccgcctgaa accagaagca 780 gacaccctcc ctcgaagtgg tgagcaggaa cggaagaaccccatggcact ctatatcctg 840 aaggacaagg actccccgga gaccgaggag aacccggccccggagcctcg aagcgcgacg 900 gagcccggcc cgcccggcta ctccgtgtct cccgccgtgcccggccgctc gccggggctg 960 cccatccgct ctgcccgccg ctacccgcgc tccccagcgcgctccccagc caccggccgg 1020 acacactcgt cgccgcccag ggccccgagc tcgcccggccgctcgcgcag cgcctcgcgc 1080 acactgcgga ctgcgggcgt gcacataatc cgcgagcaagacgaggccgg cccggtggag 1140 atcagcgcct ga 1152 26 383 PRT Homo sapiens 26Val Asn Ile Thr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly Lys 1 5 1015 Ser Ala Leu Leu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Arg Pro 20 2530 Val Val Lys Trp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val Gln 35 4045 Ser Ile Gly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg Asp 50 5560 Arg Ile Arg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu Gln 65 7075 80 Leu Ala Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile Thr Asp Asp 8590 95 Thr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val Asp Val Pro Ile100 105 110 Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr Val Leu Glu LeuSer 115 120 125 Glu Ala Phe Thr Leu Asn Cys Ser His Glu Asn Gly Thr LysPro Ser 130 135 140 Tyr Thr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn AspSer Arg Met 145 150 155 160 Leu Leu Ser Pro Asp Gln Lys Val Leu Thr IleThr Arg Val Leu Met 165 170 175 Glu Asp Asp Asp Leu Tyr Ser Cys Met ValGlu Asn Pro Ile Ser Gln 180 185 190 Gly Arg Ser Leu Pro Val Lys Ile ThrVal Tyr Arg Arg Ser Ser Leu 195 200 205 Tyr Ile Ile Leu Ser Thr Gly GlyIle Phe Leu Leu Val Thr Leu Val 210 215 220 Thr Val Cys Ala Cys Trp LysPro Ser Lys Arg Lys Gln Lys Lys Leu 225 230 235 240 Glu Lys Gln Asn SerLeu Glu Tyr Met Asp Gln Asn Asp Asp Arg Leu 245 250 255 Lys Pro Glu AlaAsp Thr Leu Pro Arg Ser Gly Glu Gln Glu Arg Lys 260 265 270 Asn Pro MetAla Leu Tyr Ile Leu Lys Asp Lys Asp Ser Pro Glu Thr 275 280 285 Glu GluAsn Pro Ala Pro Glu Pro Arg Ser Ala Thr Glu Pro Gly Pro 290 295 300 ProGly Tyr Ser Val Ser Pro Ala Val Pro Gly Arg Ser Pro Gly Leu 305 310 315320 Pro Ile Arg Ser Ala Arg Arg Tyr Pro Arg Ser Pro Ala Arg Ser Pro 325330 335 Ala Thr Gly Arg Thr His Ser Ser Pro Pro Arg Ala Pro Ser Ser Pro340 345 350 Gly Arg Ser Arg Ser Ala Ser Arg Thr Leu Arg Thr Ala Gly ValHis 355 360 365 Ile Ile Arg Glu Gln Asp Glu Ala Gly Pro Val Glu Ile SerAla 370 375 380 27 37 DNA Artificial Sequence GCP Forward Primer 27ggggacaagt ttgtacaaaa aagcaggctt cgccacc 37 28 51 DNA ArtificialSequence GCP Reverse Primer 28 ggggaccact ttgtacaaga aagctgggtttcaatggtga tggtgatggt g 51 29 41 DNA Artificial SequenceINSP052-B1P-exon1F Primer 29 gcaggcttcg ccaccatgaa gagagaaagg ggagccctgtc 41 30 36 DNA Artificial Sequence INSP052-exon1R Primer 30 tcaccccctccagggggtct gtctggatca gaagaa 36 31 36 DNA Artificial SequenceINSP052-exon2F Primer 31 ttcttctgat ccagacagac cccctggagg gggtga 36 3236 DNA Artificial Sequence INSP052-exon2R Primer 32 gtggcctcgaaatgggcaca tctacagtaa ggttga 36 33 36 DNA Artificial SequenceINSP052-exon3F Primer 33 caaccttact gtagatgtgc ccatttcgag gccaca 36 3432 DNA Artificial Sequence INSP052-exon3R Primer 34 ggagcttcttctgtatacgg tgatcttgac ag 32 35 35 DNA Artificial Sequence INSP052-5HIS-RPrimer 35 gtgatggtga tggtgggagc ttcttctgta tacgg 35 36 20 DNA ArtificialSequence pEAK12-F Primer 36 gccagcttgg cacttgatgt 20 37 20 DNAArtificial Sequence pEAK12-R Primer 37 gatggaggtg gacgtgtcag 20 38 25DNA Artificial Sequence pENTR-F1 Primer 38 tcgcgttaac gctagcatgg atctc25 39 24 DNA Artificial Sequence pENTR-R1 Primer 39 gtaacatcagagattttgag acac 24 40 2024 DNA Homo sapiens 40 atgaagagag aaaggggagccctgtccaga gcctccaggg ccctgcgcct tgctcctttt 60 gtctaccttc ttctgatccagacagacccc ctggaggggg tgaacatcac cagccccgtg 120 cgcctgatcc atggcaccgtggggaagtcg gctctgcttt ctgtgcagta cagcagtacc 180 agcagcgaca ggcctgtagtgaagtggcag ctgaagcggg acaagccagt gaccgtggtg 240 cagtccattg gcacagaggtcatcggcacc ctgcggcctg actatcgaga ccgtatccga 300 ctctttgaaa atggctccctgcttctcagc gacctgcagc tggccgatga gggcacctat 360 gaggtcgaga tctccatcaccgacgacacc ttcactgggg agaagaccat caaccttact 420 gtagatgtgc ccatttcgaggccacaggtg ttggtggctt caaccactgt gctggagctc 480 agcgaggcct tcaccttgaactgctcacat gagaatggca ccaagcccag ctacacctgg 540 ctgaaggatg gcaagcccctcctcaatgac tcgagaatgc tcctgtcccc cgaccaaaag 600 gtgctcacca tcacccgcgtgctcatggag gatgacgacc tgtacagctg catggtggag 660 aaccccatca gccagggccgcagcctgcct gtcaagatca ccgtatacag aagaagctcc 720 ctttacatca tcttgtctacaggaggcatc ttcctccttg tgaccttggt gacagtctgt 780 gcctgctgga aaccctccaaaaggaaacag aagaagctag aaaagcaaaa ctccctggaa 840 tacatggatc agaatgatgaccgcctgaaa ccagaagcag acaccctccc tcgaagtggt 900 gagcaggaac ggaagaaccccatggcactc tatatcctga aggacaagga ctccccggag 960 accgaggaga acccggccccggagcctcga agcgcgacgg agcccggccc gcccggctac 1020 tccgtgtctc ccgccgtgcccggccgctcg ccggggctgc ccatccgctc tgcccgccgc 1080 tacccgcgct ccccagcgcgctccccagcc accggccgga cacactcgtc gccgcccagg 1140 gccccgagct cgcccggccgctcgcgcagc gcctcgcgca cactgcggac tgcgggcgtg 1200 cacataatcc gcgagcaagacgaggccggc ccggtggaga tcagcgcctg agccgcctcg 1260 gatcccctga gaggcgcccgcggtctgcgg ccagtggccc gggggaaagc tggggctggg 1320 aagcccgggc gcggcgcgctggggacgagg ggaggtcccg ggggggcgct ggtgtctcgg 1380 gtgtgaacgt gtatgagcatgcgcagacgg aggcgggtgc gcggaggcgg cagtgttgat 1440 atggtgaaac cgggtcgcatttgcttccgg tttactggct gtgtcctcac ttggtatagg 1500 ttgtgccctc ttaggaccacatagattatt acatttctgg cccaataccc aaaagggttt 1560 tatggaaact aacatcagtaacctaacccc cgtgactatc ctgtgctctt cctagggagc 1620 tgtgttgttt cccacccaccacccttccct ctgaacaaat gcctgagtgc tggggcactt 1680 tttttttttt tttttttttttttttttttg caagttcaga ttagagaggc cactttccca 1740 gaatccacag ctgcactaagctaaggagaa gccagatgcc ggttactggg tgtgcagggg 1800 ctgttctgag ctggggggatcattgtgaag gccttcttcc ctgggcacct ggtacctggg 1860 gacctacaag gtggtgagggaagggtacga gtacattcct tttccctctg acctgggcgc 1920 tagcaagggc aaagaacccgagcctgccag cttggcctcc tcccacagcc tccctcggag 1980 gcatgccatg ccaagcactctttctgtctc tgttcatgaa taaa 2024 41 416 PRT Homo sapiens 41 Met Lys ArgGlu Arg Gly Ala Leu Ser Arg Ala Ser Arg Ala Leu Arg 1 5 10 15 Leu AlaPro Phe Val Tyr Leu Leu Leu Ile Gln Thr Asp Pro Leu Glu 20 25 30 Gly ValAsn Ile Thr Ser Pro Val Arg Leu Ile His Gly Thr Val Gly 35 40 45 Lys SerAla Leu Leu Ser Val Gln Tyr Ser Ser Thr Ser Ser Asp Arg 50 55 60 Pro ValVal Lys Trp Gln Leu Lys Arg Asp Lys Pro Val Thr Val Val 65 70 75 80 GlnSer Ile Gly Thr Glu Val Ile Gly Thr Leu Arg Pro Asp Tyr Arg 85 90 95 AspArg Ile Arg Leu Phe Glu Asn Gly Ser Leu Leu Leu Ser Asp Leu 100 105 110Gln Leu Ala Asp Glu Gly Thr Tyr Glu Val Glu Ile Ser Ile Thr Asp 115 120125 Asp Thr Phe Thr Gly Glu Lys Thr Ile Asn Leu Thr Val Asp Val Pro 130135 140 Ile Ser Arg Pro Gln Val Leu Val Ala Ser Thr Thr Val Leu Glu Leu145 150 155 160 Ser Glu Ala Phe Thr Leu Asn Cys Ser His Glu Asn Gly ThrLys Pro 165 170 175 Ser Tyr Thr Trp Leu Lys Asp Gly Lys Pro Leu Leu AsnAsp Ser Arg 180 185 190 Met Leu Leu Ser Pro Asp Gln Lys Val Leu Thr IleThr Arg Val Leu 195 200 205 Met Glu Asp Asp Asp Leu Tyr Ser Cys Met ValGlu Asn Pro Ile Ser 210 215 220 Gln Gly Arg Ser Leu Pro Val Lys Ile ThrVal Tyr Arg Arg Ser Ser 225 230 235 240 Leu Tyr Ile Ile Leu Ser Thr GlyGly Ile Phe Leu Leu Val Thr Leu 245 250 255 Val Thr Val Cys Ala Cys TrpLys Pro Ser Lys Arg Lys Gln Lys Lys 260 265 270 Leu Glu Lys Gln Asn SerLeu Glu Tyr Met Asp Gln Asn Asp Asp Arg 275 280 285 Leu Lys Pro Glu AlaAsp Thr Leu Pro Arg Ser Gly Glu Gln Glu Arg 290 295 300 Lys Asn Pro MetAla Leu Tyr Ile Leu Lys Asp Lys Asp Ser Pro Glu 305 310 315 320 Thr GluGlu Asn Pro Ala Pro Glu Pro Arg Ser Ala Thr Glu Pro Gly 325 330 335 ProPro Gly Tyr Ser Val Ser Pro Ala Val Pro Gly Arg Ser Pro Gly 340 345 350Leu Pro Ile Arg Ser Ala Arg Arg Tyr Pro Arg Ser Pro Ala Arg Ser 355 360365 Pro Ala Thr Gly Arg Thr His Ser Ser Pro Pro Arg Ala Pro Ser Ser 370375 380 Pro Gly Arg Ser Arg Ser Ala Ser Arg Thr Leu Arg Thr Ala Gly Val385 390 395 400 His Ile Ile Arg Glu Gln Asp Glu Ala Gly Pro Val Glu IleSer Ala 405 410 415 42 860 DNA Homo sapiens 42 acaagtttgt acaaaaaagcaggcttcgcc accatgaaga gagaaagggg agccctgtcc 60 agagcctcca gggccctgcgccttgctcct tttgtctacc ttcttctgat ccagacagac 120 cccctggagg gggtgaacatcaccagcccc gtgcgcctga tccatggcac cgtggggaag 180 tcggctctgc tttctgtgcagtacagcagt accagcagcg acaggcctgt agtgaagtgg 240 cagctgaagc gggacaagccagtgaccgtg gtgcagtcca ttggcacaga ggtcatcggc 300 accctgcggc ctgactatcgagaccgtatc cgactctttg aaaatggctc cctgcttctc 360 agcgacctgc agctggccgatgagggcacc tatgaggtcg agatctccat caccgacgac 420 accttcactg gggagaagaccatcaacctt actgtagatg tgcccatttc gaggccacag 480 accttcactg gggagaagaccatcaacctt actgtagatg tgcccatttc gaggccacag 540 gtgttggtgg cttcaaccactgtgctggag ctcagcgagg ccttcacctt gaactgctca 600 catgagaatg gcaccaagcccagctacacc tggctgaagg atggcaagcc cctcctcaat 660 gactcgagaa tgctcctgtcccccgaccaa aaggtgctca ccatcacccg cgtgctcatg 720 gaggatgacg acctgtacagctgcatggtg gagaacccca tcagccaggg ccgcagcctg 780 cctgtcaaga tcaccgtatacagaagaagc tcccaccatc accatcacca ttgaaaccca 840 gctttcttgt acaaagtggt860 43 246 PRT Homo sapiens 43 Met Lys Arg Glu Arg Gly Ala Leu Ser ArgAla Ser Arg Ala Leu Arg 1 5 10 15 Leu Ala Pro Phe Val Tyr Leu Leu LeuIle Gln Thr Asp Pro Leu Glu 20 25 30 Gly Val Asn Ile Thr Ser Pro Val ArgLeu Ile His Gly Thr Val Gly 35 40 45 Lys Ser Ala Leu Leu Ser Val Gln TyrSer Ser Thr Ser Ser Asp Arg 50 55 60 Pro Val Val Lys Trp Gln Leu Lys ArgAsp Lys Pro Val Thr Val Val 65 70 75 80 Gln Ser Ile Gly Thr Glu Val IleGly Thr Leu Arg Pro Asp Tyr Arg 85 90 95 Asp Arg Ile Arg Leu Phe Glu AsnGly Ser Leu Leu Leu Ser Asp Leu 100 105 110 Gln Leu Ala Asp Glu Gly ThrTyr Glu Val Glu Ile Ser Ile Thr Asp 115 120 125 Asp Thr Phe Thr Gly GluLys Thr Ile Asn Leu Thr Val Asp Val Pro 130 135 140 Ile Ser Arg Pro GlnVal Leu Val Ala Ser Thr Thr Val Leu Glu Leu 145 150 155 160 Ser Glu AlaPhe Thr Leu Asn Cys Ser His Glu Asn Gly Thr Lys Pro 165 170 175 Ser TyrThr Trp Leu Lys Asp Gly Lys Pro Leu Leu Asn Asp Ser Arg 180 185 190 MetLeu Leu Ser Pro Asp Gln Lys Val Leu Thr Ile Thr Arg Val Leu 195 200 205Met Glu Asp Asp Asp Leu Tyr Ser Cys Met Val Glu Asn Pro Ile Ser 210 215220 Gln Gly Arg Ser Leu Pro Val Lys Ile Thr Val Tyr Arg Arg Ser Ser 225230 235 240 His His His His His His 245

1. A polypeptide, which polypeptide: i. comprises or consists of theamino acid sequence as recited in SEQ ID NO: 16 or SEQ ID NO:26; or ii.is a fragment of a polypeptide which comprises or consists of the aminoacid sequence as recited in SEQ. ID NO: 16 or SEQ ID NO:26, having theactivity of a polypeptide according to (i), or having an antigenicdeterminant in common with a polypeptide according to (i); or iii. is afunctional equivalent of (i) or (ii).
 2. A polypeptide according toclaim 1 part ii) which comprises or consists of the amino acid sequenceas recited in SEQ ID NO:20 or in SEQ ID NO:22.
 3. A polypeptide which isa functional equivalent according to claim 1 (iii), characterised inthat it is homologous to the amino acid sequence as recited in SEQ IDNO: 16 or SEQ ID NO:26 and has activity as an antagonist of cytokineexpression and/or secretion.
 4. A purified nucleic acid molecule whichencodes a polypeptide according to claim
 1. 5. A purified nucleic acidmolecule according to claim 4, which comprises the nucleic acid sequenceas recited in SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:25, oris a redundant equivalent or fragment thereof.
 6. A purified nucleicacid molecule according to claim 5, which consists of the nucleic acidsequence as recited in SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21 or SEQID NO:25.
 7. A purified nucleic acid molecule which hybridizes underhigh stringency conditions with a nucleic acid molecule according toclaim
 4. 8. A vector comprising a nucleic acid molecule as recited inclaim
 4. 9. A host cell transformed with a vector according to claim 8.10. A ligand which binds specifically to, and which preferably inhibitsthe activity of a polypeptide according to claim
 1. 11. A ligandaccording to claim 10, which is an antibody.
 12. A compound that eitherincreases or decreases the level of expression or activity of apolypeptide according to claim
 1. 13. A compound that either increasesor decreases the level of expression or activity of a polypeptideaccording to claim 1, that binds to a polypeptide according to claim 1without inducing any of the biological effects of the polypeptide.
 14. Acompound according to claim 13, which is a natural or modifiedsubstrate, ligand, enzyme, receptor or structural or functional mimetic.15. A polypeptide according to claim 1, a nucleic acid moleculeaccording to claim 4, a vector according to claim 8, a host cellaccording to claim 9, a ligand according to claim 10, or a compoundaccording to claim 12, for use in therapy or diagnosis of disease.
 16. Apolypeptide according to claim 1 for use in therapy or diagnosis ofdisease.
 17. A nucleic acid molecule according to claim 4 for use intherapy or diagnosis of disease.
 18. A vector according to claim 8 foruse in therapy or diagnosis of disease.
 19. A host cell according toclaim 9 for use in therapy or diagnosis of disease.
 20. A ligandaccording to claim 10 for use in therapy or diagnosis of disease.
 21. Acompound according to claim 12 for use in therapy or diagnosis ofdisease.
 22. A method of diagnosing a disease in a patient, comprisingassessing the level of expression of a natural gene encoding apolypeptide according to claim 1, or assessing the activity of apolypeptide according to claim 1, in tissue from said patient andcomparing said level of expression or activity to a control level,wherein a level that is different to said control level is indicative ofdisease.
 23. A method according to claim 22 that is carried out invitro.
 24. A method of diagnosing a disease in a patient, comprisingassessing the level of expression of a natural gene encoding apolypeptide according to claim 1, or assessing the activity of apolypeptide according to claim 1, in tissue from said patient andcomparing said level of expression or activity to a control level,wherein a level that is different to said control level is indicative ofdisease, which comprises the steps of: (a) contacting a ligand whichbinds specifically to, and which preferably inhibits the activity of apolypeptide according to claim 1 with a biological sample underconditions suitable for the formation of a ligand-polypeptide complex;and (b) detecting said complex.
 25. A method of diagnosing a disease ina patient, comprising assessing the level of expression of a naturalgene encoding a polypeptide according to claim 1, or assessing theactivity of a polypeptide according to claim 1, in tissue from saidpatient and comparing said level of expression or activity to a controllevel, wherein a level that is different to said control level isindicative of disease, comprising the steps of: a) contacting a sampleof tissue from the patient with a nucleic acid probe under stringentconditions that allow the formation of a hybrid complex between anucleic acid molecule which encodes a polypeptide according to claim 1and the probe; b) contacting a control sample with said probe under thesame conditions used in step a); and c) detecting the presence of hybridcomplexes in said samples; wherein detection of levels of the hybridcomplex in the patient sample that differ from levels of the hybridcomplex in the control sample is indicative of disease.
 26. A method ofdiagnosing a disease in a patient, comprising assessing the level ofexpression of a natural gene encoding a polypeptide according to claim1, or assessing the activity of a polypeptide according to claim 1, intissue from said patient and comparing said level of expression oractivity to a control level, wherein a level that is different to saidcontrol level is indicative of disease, that is carried out in vitro,comprising: a) contacting a sample of nucleic acid from tissue of thepatient with a nucleic acid primer under stringent conditions that allowthe formation of a hybrid complex between a nucleic acid molecule whichencodes a polypeptide according to claim 1 and the primer; b) contactinga control sample with said primer under the same conditions used in stepa); and c) amplifying the sampled nucleic acid; and d) detecting thelevel of amplified nucleic acid from both patient and control samples;wherein detection of levels of the amplified nucleic acid in the patientsample that differ significantly from levels of the amplified nucleicacid in the control sample is indicative of disease.
 27. A method ofdiagnosing a disease in a patient, comprising assessing the level ofexpression of a natural gene encoding a polypeptide according to claim1, or assessing the activity of a polypeptide according to claim 1, intissue from said patient and comparing said level of expression oractivity to a control level, wherein a level that is different to saidcontrol level is indicative of disease, that is carried out in vitro,comprising: a) obtaining a tissue sample from a patient being tested fordisease; b) isolating a nucleic acid molecule which encodes apolypeptide according to claim 1 from said tissue sample; and c)diagnosing the patient for disease by detecting the presence of amutation which is associated with disease in the nucleic acid moleculeas an indication of the disease.
 28. The method of claim 27, furthercomprising amplifying the nucleic acid molecule to form an amplifiedproduct and detecting the presence or absence of a mutation in theamplified product.
 29. The method of claim 27, wherein the presence orabsence of the mutation in the patient is detected by contacting saidnucleic acid molecule with a nucleic acid probe that hybridises to saidnucleic acid molecule under stringent conditions to form a hybriddouble-stranded molecule, the hybrid double-stranded molecule having anunhybridised portion of the nucleic acid probe strand at any portioncorresponding to a mutation associated with disease; and detecting thepresence or absence of an unhybridised portion of the probe strand as anindication of the presence or absence of a disease-associated mutation.30. A method according to claim 22, wherein said disease is anauto-immune, viral or acute liver disease, including alcoholic liverfailure, or inflammatory disease.
 31. A method of using a polypeptideaccording to claim 1 as an antagonist of cytokine expression and/orsecretion.
 32. A pharmaceutical composition comprising a polypeptideaccording to claim
 1. 33. A pharmaceutical composition comprising anucleic acid molecule according to claim
 4. 34. A pharmaceuticalcomposition comprising a vector according to claim
 8. 35. Apharmaceutical composition comprising a host cell according to claim 9.36. A pharmaceutical composition comprising a ligand according to claim10.
 37. A pharmaceutical composition comprising a compound accordingclaim
 12. 38. A vaccine composition comprising a polypeptide accordingto claim
 1. 39. A vaccine composition comprising a nucleic acid moleculeaccording to claim
 4. 40. A method of using a polypeptide according toclaim 1 in the manufacture of a medicament for the treatment of anauto-immune disease, viral or acute liver disease, including alcoholicliver failure, or inflammatory disease.
 41. A method of using nucleicacid molecule according to claim 4 in the manufacture of a medicamentfor the treatment of an auto-immune disease, viral or acute liverdisease, including alcoholic liver failure, or inflammatory disease. 42.A method of using a vector according to claim 8 in the manufacture of amedicament for the treatment of an auto-immune disease, viral or acuteliver disease, including alcoholic liver failure, or inflammatorydisease.
 43. A method of using a host cell according to claim 9, in themanufacture of a medicament for the treatment of an auto-immune disease,viral or acute liver disease, including alcoholic liver failure, orinflammatory disease.
 44. A method of using a ligand according to claim10 in the manufacture of a medicament for the treatment of anauto-immune disease, viral or acute liver disease, including alcoholicliver failure, or inflammatory disease.
 45. A method of using a compoundaccording to claim 12 in the manufacture of a medicament for thetreatment of an auto-immune disease, viral or acute liver disease,including alcoholic liver failure, or inflammatory disease.
 46. A methodof using a pharmaceutical composition of claim 32 in the manufacture ofa medicament for the treatment of an auto-immune disease, viral or acuteliver disease, including alcoholic liver failure, or inflammatorydisease.
 47. A method of treating a disease in a patient, comprisingadministering to the patient a polypeptide according to claim
 1. 48. Amethod according to claim 47, wherein, for diseases in which theexpression of the natural gene or the activity of the polypeptide islower in a diseased patient when compared to the level of expression oractivity in a healthy patient, the polypeptide administered to thepatient is an agonist.
 49. A method according to claim 47, wherein, fordiseases in which the expression of the natural gene or activity of thepolypeptide is higher in a diseased patient when compared to the levelof expression or activity in a healthy patient, the polypeptideadministered to the patient is an antagonist.
 50. A method of treating adisease in a patient, comprising administering to the patient a nucleicacid molecule according to claim
 4. 51. A method according to claim 50,wherein, for diseases in which the expression of the natural gene or theactivity of the polypeptide is lower in a diseased patient when comparedto the level of expression or activity in a healthy patient, the nucleicacid molecule administered to the patient is an agonist.
 52. A methodaccording to claim 50, wherein, for diseases in which the expression ofthe natural gene or activity of the polypeptide is higher in a diseasedpatient when compared to the level of expression or activity in ahealthy patient, the nucleic acid molecule administered to the patientis an antagonist.
 53. A method of treating a disease in a patient,comprising administering to the patient a vector according to claim 8.54. A method according to claim 53, wherein, for diseases in which theexpression of the natural gene or the activity of the polypeptide islower in a diseased patient when compared to the level of expression oractivity in a healthy patient, the vector administered to the patient isan agonist.
 55. A method according to claim 53, wherein, for diseases inwhich the expression of the natural gene or activity of the polypeptideis higher in a diseased patient when compared to the level of expressionor activity in a healthy patient, the vector administered to the patientis an antagonist.
 56. A method of treating a disease in a patient,comprising administering to the patient a host cell according to claim9.
 57. A method according to claim 56, wherein, for diseases in whichthe expression of the natural gene or the activity of the polypeptide islower in a diseased patient when compared to the level of expression oractivity in a healthy patient, the host cell administered to the patientis an agonist.
 58. A method according to claim 56, wherein, for diseasesin which the expression of the natural gene or activity of thepolypeptide is higher in a diseased patient when compared to the levelof expression or activity in a healthy patient, the host celladministered to the patient is an antagonist.
 59. A method of treating adisease in a patient, comprising administering to the patient a ligandaccording to claim
 10. 60. A method according to claim 59, wherein, fordiseases in which the expression of the natural gene or the activity ofthe polypeptide is lower in a diseased patient when compared to thelevel of expression or activity in a healthy patient, the ligandadministered to the patient is an agonist.
 61. A method according toclaim 59, wherein, for diseases in which the expression of the naturalgene or activity of the polypeptide is higher in a diseased patient whencompared to the level of expression or activity in a healthy patient,the ligand administered to the patient is an antagonist.
 62. A method oftreating a disease in a patient, comprising administering to the patienta compound according to claim
 12. 63. A method according to claim 62,wherein, for diseases in which the expression of the natural gene or theactivity of the polypeptide is lower in a diseased patient when comparedto the level of expression or activity in a healthy patient, thecompound administered to the patient is an agonist.
 64. A methodaccording to claim 62, wherein, for diseases in which the expression ofthe natural gene or activity of the polypeptide is higher in a diseasedpatient when compared to the level of expression or activity in ahealthy patient, the compound administered to the patient is anantagonist.
 65. A method of treating a disease in a patient, comprisingadministering to the patient a pharmaceutical composition of claim 66.66. A method according to claim 65, wherein, for diseases in which theexpression of the natural gene or the activity of the polypeptide islower in a diseased patient when compared to the level of expression oractivity in a healthy patient, the composition administered to thepatient is an agonist.
 67. A method according to claim 65, wherein, fordiseases in which the expression of the natural gene or activity of thepolypeptide is higher in a diseased patient when compared to the levelof expression or activity in a healthy patient, the compositionadministered to the patient is an antagonist.
 68. A method of monitoringthe therapeutic treatment of disease in a patient, comprising monitoringover a period of time the level of expression or activity of apolypeptide according to claim 1 in tissue from said patient, whereinaltering said level of expression or activity over the period of timetowards a control level is indicative of regression of said disease. 69.A method of monitoring the therapeutic treatment of disease in apatient, comprising monitoring over a period of time the level ofexpression of a nucleic acid molecule according to claim 4 in tissuefrom said patient, wherein altering said level of expression or activityover the period of time towards a control level is indicative ofregression of said disease.
 70. A method for the identification of acompound that is effective in the treatment and/or diagnosis of disease,comprising contacting a polypeptide according to claim 1 with one ormore compounds suspected of possessing binding affinity for saidpolypeptide or nucleic acid molecule, and selecting a compound thatbinds specifically to said nucleic acid molecule or polypeptide.
 71. Amethod for the identification of a compound that is effective in thetreatment and/or diagnosis of disease, comprising contacting a nucleicacid molecule according to claim 4 with one or more compounds suspectedof possessing binding affinity for said polypeptide or nucleic acidmolecule, and selecting a compound that binds specifically to saidnucleic acid molecule or polypeptide.
 72. A kit useful for diagnosingdisease comprising a first container containing a nucleic acid probethat hybridises under stringent conditions with a nucleic acid moleculeaccording to claim 4; a second container containing primers useful foramplifying said nucleic acid molecule; and instructions for using theprobe and primers for facilitating the diagnosis of disease.
 73. The kitof claim 72, further comprising a third container holding an agent fordigesting unhybridised RNA.
 74. A kit comprising an array of nucleicacid molecules, at least one of which is a nucleic acid moleculeaccording to claim
 4. 75. A kit comprising one or more antibodies thatbind to a polypeptide as recited in claim 1, and a reagent useful forthe detection of a binding reaction between said antibody and saidpolypeptide.
 76. A transgenic or knockout non-human animal that has beentransformed to express higher, lower or absent levels of a polypeptideaccording to claim
 1. 77. A method for screening for a compoundeffective to treat disease, by contacting a non-human transgenic animalaccording to claim 76 with a candidate compound and determining theeffect of the compound on the disease of the animal.